Stec et al. Zoological Letters (2021) 7:9 https://doi.org/10.1186/s40851-021-00176-w

RESEARCH ARTICLE Open Access Integrative resolves identities within the pallarii complex (Eutardigrada: ) Daniel Stec1*† , Matteo Vecchi2*†, Magdalena Dudziak1, Paul J. Bartels3, Sara Calhim2 and Łukasz Michalczyk1†

Abstract The taxonomy of many groups of meiofauna is challenging due to their low number of diagnostic morphological characters and their small body size. Therefore, with the advent of molecular techniques that provide a new source of traits, many cryptic species have started to be discovered. are not an exception, and many once thought to be cosmopolitan taxa are being found to be complexes of phenotypically similar species. Macrobiotus pallarii Maucci, 1954 was originally described in South Italy and has been subsequently recorded in Europe, America, and Asia. This allegedly wide geographic range suggests that multiple species may be hidden under this name. Moreover, recently, genetic evidence to support this was put forward, and the Macrobiotus pallarii complex has been proposed to accommodate putative species related to M. pallarii. Here, we describe three new pseudocryptic species based on populations that would have been all classified as Macrobiotus pallarii if molecular methods were not employed. Using an integrative taxonomy approach, we analyzed and eggs from the topotypic population of Macrobiotus pallarii, together with four other populations of the complex. We recovered four distinct phylogenetic lineages that, despite the overlap of morphometric traits, can be separated phenotypically by subtle but discrete morphological characters. One lineage corresponds to Macrobiotus pallarii, whereas the other three are newly described as Macrobiotus margoae Stec, Vecchi & Bartels, sp. nov.fromtheUSA,Macrobiotus ripperi Stec, Vecchi & Michalczyk, sp. nov. from Poland and Finland, and Macrobiotus pseudopallarii Stec, Vecchi & Michalczyk, sp. nov.fromMontenegro.To facilitate species identification, we provide a dichotomous key for species of the M. pallarii complex. Delimitation of these pseudocryptic taxa highlights the need for an integrative approach to uncover the phylum’s diversity in full. Keywords: cryptic taxa, DNA barcoding, egg ornamentation, new species, species complex, species delimitation

* Correspondence: [email protected]; [email protected] †Daniel Stec and Matteo Vecchi contributed equally to this work. †Łukasz Michalczyk is the senior author. 1Department of Invertebrate Evolution, Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University, Gronostajowa 9, 30-387 Kraków, Poland 2Department of Biological and Environmental Science, University of Jyväskylä, PO Box 35, FI-40014 Jyväskylä, Finland Full list of author information is available at the end of the article

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Background described from South Italy and a senior synonym of Tardigrades constitute a phylum of microinvertebrates Macrobiotus aviglianae Robotti, 1970 [23, 24], which inhabiting both terrestrial and aquatic (freshwater and was described from North Italy. Macrobiotus pallarii marine) habitats worldwide [1], with approximately has been found in various localities in Europe, Asia 1300 species known so far [2–4]. tax- and North America [25]; however, none of the re- onomy is challenging due to their small size, limited cords outside the type locality have been genetically number of taxonomically informative traits, and many verified; thus, the true geographic range of this spe- outdated species descriptions that do not comply with cies is unknown. The second species, Macrobiotus modern taxonomic standards. Advances have been ragonesei Binda et al., 2001 [26], was described and made possible with the recent advent of integrative found only in Central Africa [26, 27], whereas the taxonomy (e.g., [5–10]) that includes the use of DNA third species, Macrobiotus caymanensis Meyer, 2011 sequencing with detailed morphological techniques [28], was found only in the Cayman Islands (the such as scanning electron microscopy (SEM) and so- Caribbean). Additionally, three phylogenetically dis- phisticated analysis of morphometric data. Many stud- tinct but undescribed and morphologically very simi- ies using an integrative taxonomy approach have lar species of this group have been recently detected recently revealed that various tardigrade species once in several localities in Europe and North America thought to be widespread are actually complexes of [19]. Given that the existence of these species and cryptic species with more localized distributions [5, 6, morphological characters that allow for their pheno- 11, 12]. In addition to their distribution, tardigrade typic identification became apparent only after the cryptic species have also been shown to diverge based molecular analysis had been performed, they can be on reproductive mode [13], ploidy [5], and anhydro- considered pseudocryptic species [29, 30]. To eluci- biotic survival [14]. Three classes have been recog- date the taxonomy of this species complex, we ana- nized in the phylum Tardigrada: , lyzed (and resequenced) the populations reported by Mesotardigrada, and Eutardigrada [15]. The class Het- Stec et al. [19], as well as individuals and eggs from erotardigrada encompasses both primarily marine tar- thetypelocalityofM. pallarii, from phylogenetic digrades (Arthrotardigrada) and mostly (multilocus phylogeny and species delimitation), mor- limnoterrestrial armoured tardigrades (). phometric (principal component analysis; PCA), and The class Mesotardigrada has been found only once morphological perspectives. Morphometric traits alone from a thermal spring in Japan and is considered a do not allow for a separation of M. pallarii and the classis dubium [16]. The class Eutardigrada comprises three new pseudocryptic species, as almost all their two mostly limnoterrestrial orders, Apochela and biometric values overlap considerably. However, our . The order Parachela, which is the most analysis identified some qualitative phenotypic charac- common, widespread, and speciose group with a wide ters that could be used to separate and formally de- range of dietary preferences [17], comprises the scribe three new species within this morphologically superfamily Macrobiotoidea. The nominal for uniform species complex. Last, to facilitate species the superfamily Macrobiotus CAS Schultze, 1834 [18] identification, we provide a dichotomous key for spe- is characterized by the presence of symmetrical cies of the M. pallarii complex. diploclaws, a rigid buccal tube with a straight ventral bar lacking a ventral hook, two macroplacoids and one microplacoid in the pharynx, 10 peribuccal lamel- Materials and methods lae, pores in the cuticle, and freely laid ornamented Samples and specimens eggs [19]. Macrobiotus is one of the most species-rich Five populations of the Macrobiotus pallarii complex and widespread genera in the phylum, and it was also were isolated from moss samples collected from five lo- the first formally described tardigrade genus [20]. An- calities in Europe and North America (see Table 1 for imals of the Macrobiotus pallarii complex have the details). The samples were examined for tardigrades very typical morphology of Macrobiotus. However, using the protocol by [31], with modifications described this group is characterized by egg ornamentation in detail in [32]. The live animals and eggs were placed composed of large conical processes separated by a into culture. Specimens were reared in plastic Petri single row of areolation (such eggs are also known in dishes according to the protocol by [32]. To perform the other macrobiotid genera, such as Paramacrobiotus taxonomic analysis of these species/populations, animals Guidetti et al., 2009 [21] but have not been found in and eggs were taken from the cultures and split into sev- other Macrobiotus species). To date, three described eral groups for specific analyses: (i) morphological and species belong to this complex, with the type species morphometric analyses with light contrast microscopy being Macrobiotus pallarii Maucci, 1954 [22], (LCM), (ii) morphological analysis with SEM, and (iii) Stec et al. Zoological Letters (2021) 7:9 Page 3 of 45

Table 1 Information on moss samples with the populations of the Macrobiotus pallarii complex analyzed in the present study. *Type locality of Macrobiotus pallarii Sample/population code Locality Coordinates and altitude Collectors FI.066 Finland, Jyväskylä, Graniitti 62°13′24.6″N Matteo Vecchi 25°46′20.4″E 84 m asl ME.007 Montenegro, Crkvine 42°47′57.54″N Aleksandra Rysiewska 19°27′18.47″E 1015 m asl PL.015 Poland, Malinówka, Yew Reserve 49°42′09″N Piotr Gąsiorek 21°55′53″E 382 m asl US.057 USA, Great Smoky Mountains 35°35′7.84″N Nate Gross & Mackenzie McClay National Park, Purchase Knob 83°4′26.47″W 1492 m asl IT.337* Italy, Cosenza, Silvana Mansio 39°18′34.5″N 16°32′19.9″E Francesco Squillace 1436 m asl

DNA sequencing (for details see sections “Material ex- Genotyping amined” provided below for each description). DNA was extracted from individual animals following a Chelex® 100 resin (BioRad) extraction method by [33] with modifications described in detail in [34]. Individuals Comparative materials from the same populations that were sequenced by Stec Measurements of the type series of M. caymanensis et al.[19] (populations FI.066-PL.015-ME.007-US.057) from the Cayman Islands (KY) and additional infor- were resequenced in the present study, as [19] provided mation on morphological traits were kindly provided sequences at the haplotype level, whereas species delimi- by Harry Meyer (McNeese State University, USA). tation methods require sequences at the level of individ- Photomicrographs of the animals and eggs from the uals. Each specimen was mounted in water and type series of M. pallarii from the Maucci collection examined under LCM prior to DNA extraction. We se- (Civic Museum of Natural History of Verona, Italy) quenced four DNA fragments, three nuclear fragments were taken by MV and Roberto Guidetti (University (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial of Modena and Reggio Emilia, Italy). Moreover, sev- fragment (COI). All fragments were amplified and se- eral eggs of a Macrobiotus polyopus group species quenced according to the protocols described in [34]; collected in Papua New Guinea (ca. 8°20′S, 146°16′E, primers with original references are listed in Table 2. 10 m asl) in 2007 by Anna Millard (University of East Sequencing products were read with an ABI 3130xl se- Anglia, UK) were compared with the Macrobiotus quencer at the Molecular Ecology Lab, Institute of En- pallarii complex populations analyzed herein. vironmental Sciences of Jagiellonian University, Kraków,

Table 2 Primers with their original references used for amplification of the four DNA fragments sequenced in the study. The primer set LCO1490-JJ + HCO2198-JJ was used for COI amplification in four populations (FI. 066, IT. 337, PL. 015, US. 057), whereas LCO1490 + HCOoutout was used for one population (ME.007) DNA marker Primer name and source Primer direction Primer sequence (5’-3’) 18S rRNA 18S_Tar_Ff1 [35] forward AGGCGAAACCGCGAATGGCTC 18S_Tar_Rr1 [35] reverse GCCGCAGGCTCCACTCCTGG 28S rRNA 28S_Eutar_F [36] forward ACCCGCTGAACTTAAGCATAT 28SR0990 [37] reverse CCTTGGTCCGTGTTTCAAGAC ITS-2 ITS2_Eutar_Ff [38] forward CGTAACGTGAATTGCAGGAC ITS2_Eutar_Rr [38] reverse TCCTCCGCTTATTGATATGC COI LCO1490-JJ [39] forward CHACWAAYCATAAAGATATYGG HCO2198-JJ [39] reverse AWACTTCVGGRTGVCCAAARAATCA LCO1490 [40] forward GGTCAACAAATCATAAAGATATTGG HCOoutout [41] reverse GTAAATATATGRTGDGCTC Stec et al. Zoological Letters (2021) 7:9 Page 4 of 45

Table 3 GenBank accession numbers of sequences used in the present study. Newly generated sequences are in bold Taxon Individual 18S 28S COI ITS2 Macrobiotus caelestis MK737073 MK737071 MK737922 MK737072 Macrobiotus pallarii complex FI.066.1 MT809075 MT809088 MT807929 FI.066.2 MT809076 MT809089 MT807930 MT809103 FI.066.3 MT807931 MT809104 FI.066.4 MT807932 MT809105 IT.337.1 MT809069 MT809081 MT807924 MT809094 IT.337.2 MT809070 MT809082 MT807925 MT809095 IT.337.3 MT809071 MT809083 MT807926 MT809096 ME.007.1 MT809065 MT809077 MT809090 ME.007.2 MT809066 MT809078 MT807920 ME.007.3 MT809067 MT809079 MT807921 MT809091 ME.007.4 MT809068 MT809080 MT807922 MT809092 ME.007.5 MT807923 MT809093 PL.015.1 MT809074 MT809086 MT809100 PL.015.2 MT809087 MT807933 MT809101 PL.015.3 MT807934 PL.015.4 MT807935 MT809102 US.057.1 MT809072 MT809084 MT807927 MT809098 US.057.2 MT809073 MT809085 US.057.3 MT807928 MT809099 US.057.4 MT809097

Poland. Sequences were processed in BioEdit ver. 7.2.5 [49]. Aligned sequences were concatenated with an in-house [42] and submitted to NCBI GenBank [43]. For acces- R script (written by MV, available upon request to the au- sion numbers see Table 3. thor). Model selection and phylogenetic reconstructions were performed on the CIPRES Science Gateway [50]. Model se- Phylogenetic analysis lection was performed for each alignment partition (6 in To reconstruct the phylogeny, we used sequences represent- total: 18S, 28S, ITS-2, and three COI codons) with Partition- ing four markers (18S rRNA, 28S rRNA, ITS-2, and COI) Finder ver. 2 [51]. Bayesian inference (BI) phylogenetic re- from five different populations of the Macrobiotus pallarii construction was performed with MrBayes ver. 3.2.6 [52] complex. Sequences were downloaded from GenBank or without BEAGLE. Four runs with one cold chain and three produced de novo (Table 3). Type sequences of Macrobiotus heated chains were run for 20 million generations with a caelestis Coughlan, Michalczyk & Stec, 2019 [44]wereused 10% burn-in, sampling a tree every 10000 generations. Pos- as the outgroup. 18S rRNA, 28S rRNA, and ITS-2 sequences terior distribution was checked with Tracer ver. 1.7 [53]. The were aligned with MAFFT ver. 7 [45, 46]withtheG-INS-i MrBayes input file is available as Supplementary Material method (thread=4, threadtb=5, threadit=0, reorder, adjustdir- (SM.02).ThephylogenetictreewasvisualizedwithFigTree ection, anysymbol, maxiterate=1000, retree 1, globalpair in- ver. 1.4.4 [54], and the image was edited with Inkscape ver. put). COI sequences were aligned according to their amino 0.92.3 [55]. acid sequences (translated with the invertebrate mitochon- drial code) with the MUSCLE algorithm [47]inMEGAver. Species delimitation 7.0.26 [48] with default settings (all gap penalties=0, max iter- Following the suggestion of [56], species delimitation ations=8, clustering method=UPGMB, lambda=24) and then was performed with both a tree-based (bPTP) and a translated back to nucleotide sequences. Alignments were distance-based (ABGD) method. Tree-based species de- visually inspected and trimmed in MEGA ver. 7.0.26 [48]. limitation was performed with bPTP software [57]on The absence of saturation in the COI and ITS-2 alignments both ITS-2 and COI trees. Single gene alignments and was confirmed with transition/transversion and saturation BI trees were produced as described above (see Phylo- plots (SM.01)producedwiththeRpackage“ape ver. 5.0” genetic Analysis section). One thousand trees were Stec et al. Zoological Letters (2021) 7:9 Page 5 of 45

sampled randomly from the posterior tree distribution Macroplacoid length sequence is given according to after discarding the burn in (250 from each chain) and [63]. Buccal tube length and the level of the stylet sup- used as input for bPTP with 100,000 generations, a thin- port insertion point were measured according to [64]. ning of 100 generations and 10% burn in. Distance- The pt index is the ratio of the length of a given struc- based species delineation was performed with the ABGD ture to the length of the buccal tube expressed as a ratio online server [58] on both ITS-2 and COI alignments [64]. Measurements of buccal tube widths, heights of obtained for the phylogenetic analysis as described claws and eggs follow [62]. Morphometric data were above. For both markers, simple distance was used, with handled using the “Parachela” ver. 1.7 template available 10 steps, a relative gap width of 1.5, and 20 bins for dis- from the Tardigrada Register [65]. Tardigrade taxonomy tance distribution. For COI, Pmin and Pmax were 0.01 followed [19, 66]. and 0.1, respectively, while for ITS-2, they were 0.0001 Morphometric data for eggs and animals were analyzed and 0.1, respectively, to accommodate the difference in with PCA. All analyses were performed with the base R divergence between these loci. software package [67]. For eggs, absolute values (raw mea- surements in μm) were used for the analysis, whereas for The p-distances the animals, relative (pt) values were analyzed. Missing As the species of the M. pallarii complex are phylogenet- data in the data set were imputed with the PCA ically and morphologically distinct [19], the p-distances imputation technique with the “imputePCA” function of for the genetic differential diagnosis were calculated the R package “missMDA ver. 1.17” [68]. The number of between species of the M. pallarii complex for the four se- components used to impute the missing values was deter- quenced markers (18S, 28S, ITS2, and COI) using the mined by cross-validation (function “estim_ncpPCA”). alignment used for the phylogenetic analysis. Pairwise dis- PCAs were performed on the scaled data with the PCA tances were calculated with the software MEGA ver. function of the package “FactoMineR ver. 2.3” [69]. PCAs 7.0.26 [48] using pairwise deletion for the Gap/Missing were visualized with the packages “ggplot2 ver. 3.3.2”, Data Treatment option. Detailed p-distance tables are “plyr ver. 1.8.6” and “gridExtra ver. 2.3” [70, 71]. The pres- provided in SM.03. ence of a structure in the PCA data was tested with a randomization procedure according to [72] on the eigen- Microscopy and imaging values and with the statistics ψ and ϕ using an in-house R Specimens for LCM were mounted on microscope slides script (written by MV, in Supplementary SM.04a-4b). in a small drop of Hoyer’s medium and secured with a PERMANOVA was performed on the PCs using the spe- cover slip, following the protocol by [59]. Slides were ex- cies hypothesis obtained by phylogenetic methods as the amined under an Olympus BX53 light microscope with independent variable with the R package “vegan ver. 2.5.6” phase (PCM) and Nomarski differential interference and “pairwiseAdonis ver. 0.3” [73]. The R script of all the contrast (NCM) associated with an Olympus DP74 morphometric analyses is available as Supplementary digital camera. To obtain clean and extended specimens SM.4a-4b. Additionally, morphometric data from the type for SEM, tardigrades were processed according to the series of Macrobiotus caymanensis were included in this protocol by [32]. Specimens were examined under high analysis (population name: Cayman). The use of Thorpe vacuum in a Versa 3D DualBeam Scanning Electron normalization proposed by [74] for the comparison of Microscope (SEM) at the ATOMIN facility of Jagiello- morphometric traits between different species was not nian University, Kraków, Poland. All figures were assem- used due to the low sample size for M. pallarii and M. bled in Corel Photo-Paint X6, ver. 16.4.1.1281. For caymanensis.Theα-level for multiple post hoc compari- structures that could not be satisfactorily focused in a sons was adjusted separately for adult and egg traits using single LCM photograph (PCM and NCM), a stack of 2– the Benjamini-Hochberg correction [75]. 6 images was taken with an equidistance of ca. 0.2 μm and assembled manually into a single deep-focus image Availability of data and materials in Corel Photo-Paint X6, ver. 16.4.1.1281. The datasets supporting the conclusions of this article are available in the GenBank repository (https://www.ncbi. Morphometrics and morphological nomenclature nlm.nih.gov/genbank; Accession Numbers in Table 3)and All measurements are given in micrometers (μm). Sam- in the Tardigrada Register: under www.tardigrada.net/ ple size was adjusted following recommendations by register/0103.htm (M. pallarii), www.tardigrada.net/ [60]. Structures were measured only if their orientation register/0104.htm (M. pseudopallarii sp. nov.), www. was suitable. Body length was measured from the anter- tardigrada.net/register/0105.htm (M. ripperi sp. nov.)and ior extremity to the end of the body, excluding the hind www.tardigrada.net/register/0106.htm (M. margoae sp. legs. The terminology used to describe oral cavity arma- nov.). New species nomenclatural acts were registered in ture and eggshell morphology follows [61, 62]. ZooBank (http://zoobank.org, see Taxonomic Account for Stec et al. Zoological Letters (2021) 7:9 Page 6 of 45

URLs). Raw morphometric measurements of the analyzed with the bPTP results (Fig. 1). For COI, only for the populations supporting the conclusions of this article are smallest prior intraspecific divergence (0.01) were six included within the article (Additional files: SM.05, species recovered (the PL and FI populations were SM.06,SM.07,SM.08,SM.09). delimited as two species); however, we did not consider this result to be valid, as it was not congruent with all Results the other nine evaluated prior intraspecific divergence Phylogenetic analysis values or with the bPTP result for the same marker. The BI phylogenetic reconstructions yielded a topology (Fig. 1) with four well-supported clades: the first clade Morphometric analysis comprised all individuals from the US population; the The randomization test in the PCA revealed that for second clade contained all individuals from the Polish both animal and egg datasets, only the first two PCs ex- (PL) and Finnish (FI) populations; the third clade con- plained more variation than expected by the null model tained individuals from the Montenegrin (ME) popula- (no data structure) (Supplementary SM.10); therefore, tion; and the fourth clade contained individuals from the only the first two PCs were retained and used for further IT population. analysis and interpretation. Additionally, the ψ and ϕ statistics of the PCA were significantly different from Species delimitation their expectations under the null model (animals: ψ= The bPTP analysis of COI and ITS-2 markers gave 132.75 p<0.001, ϕ=0.45 p<0.001; eggs: ψ=6.29 p<0.001, slightly different results (Fig. 1). The species identified ϕ=0.39 p<0.001). Principal component analysis (PCA) of based on the COI dataset are in agreement with the four the pt indices of animals (Fig. 2a) described 56% of the clades found with the phylogenetic analysis. The ITS-2 total variance with the first two components (46.0% for dataset species delimitation, however, found only three PC1 and 9.9% for PC2). PERMANOVA showed an over- species, as the IT and ME populations were delineated all significant effect of species identity on the PCs (p< as a single species. The ABGD results were congruent 0.001, Table 4). The majority of the post hoc pairwise

Fig. 1 BI Phylogenetic reconstruction of the relationships between the analyzed specimens from the 5 populations. Nodes with posterior probability (pp) <0.70 were collapsed; asterisks indicate nodes with pp=1.00. Vertical bars show the results of different species delimitation methods or discriminant characters. Horizontal colored boxes highlight the identified species described in the Taxonomic Account. A schematic representation of the dorso-caudal granulation patterns for each described species is presented (for a detailed description, see the Taxonomic Account) Stec et al. Zoological Letters (2021) 7:9 Page 7 of 45

Fig. 2 Results of PCA of animal pt indices and egg raw measurements. a Animal pt indices, 1st and 2nd Principal Components; b Egg measurements, 1st and 2nd Principal Components; Top-left quadrants: score scatterplots; Top-right and bottom-left quadrants: boxplots of single component scores; bottom-right: loading plot

PERMANOVA comparisons were significant (Table 4); pallarii + M. pseudopallarii + M. ripperi) that showed however, the species could not be separated by any of some separation in the first and second PCs (Fig. 2a). the analyzed traits (Fig. 2a), which was also indicated by According to the loading plot of PC1 and PC2 (Fig. 2a), low R2 values (Table 4), thus making morphometric in- the separation between these two groups was driven dices impractical for traditional species identification. mainly by the pt indices related to the buccal apparatus The only exception was represented by two groups of structures. Principal component analysis (PCA) of egg populations (M. margoae + M. caymanensis vs M. measurements (Fig. 2b) described 60% of the total

Table 4 Results of PERMANOVA and post hoc pairwise PERMANOVA comparisons for the first two principal components (PCs) of animal pt values; significant post hoc p-values at the α-level of p<0.045 (i.e., adjusted with the Benjamini-Hochberg correction) are in bold Term df SS F R2 p Species 4 1066.73 38.49 0.54 <0.001 Residuals 131 907.59 0.46 Total 135 1974.32 1.00 Post hoc comparisons M. pallarii vs M. ripperi sp. nov. 1 83.70 16.85 0.20 <0.001 M. ripperi sp. nov. vs M. pseudopallarii sp. nov. 1 89.40 15.00 0.14 <0.001 M. ripperi sp. nov. vs M. margoae sp. nov. 1 625.77 107.08 0.54 <0.001 M. ripperi sp. nov. vs M. caymanensis 1 166.50 36.56 0.35 <0.001 M. pseudopallarii sp. nov. vs M. margoae sp. nov. 1 754.91 82.24 0.58 <0.001 M. pseudopallarii sp. nov. vs M. caymanensis 1 270.68 30.58 0.45 <0.001 M. pallarii vs M. pseudopallarii sp. nov. 1 101.20 10.53 0.22 <0.001 M. pallarii vs M. margoae sp. nov. 1 83.99 9.00 0.20 0.002 M. pallarii vs M. caymanensis 1 51.05 5.85 0.29 0.007 M. margoae sp. nov. vs M. caymanensis 1 6.84 0.79 0.02 0.396 Stec et al. Zoological Letters (2021) 7:9 Page 8 of 45

Table 5 Results of PERMANOVA and post hoc pairwise PERMANOVA comparisons for the first two principal components (PCs) of egg measurements; significant post hoc p-values at the α-level of p<0.040 (i.e., adjusted with the Benjamini-Hochberg correction) are in bold Term df SS F R2 p Species 4 272.16 25.097 0.44 <0.001 Residuals 125 338.88 0.55 Total 129 611.03 1.00 Post hoc comparisons M. pallarii vs M. pseudopallarii sp. nov. 1 59.32 18.54 0.32 <0.001 M. pallarii vs M. margoae sp. nov. 1 91.88 43.88 0.54 <0.001 M. ripperi sp. nov. vs M. pseudopallarii sp. nov. 1 61.02 20.61 0.18 <0.001 M. ripperi sp. nov. vs M. margoae sp. nov. 1 119.54 47.97 0.35 <0.001 M. pallarii vs M. ripperi sp. nov. 1 18.26 7.42 0.09 0.002 M. caymanensis vs M. pseudopallarii sp. nov. 1 22.38 6.08 0.16 0.007 M. caymanensis vs M. margoae sp. nov. 1 11.20 4.88 0.14 0.015 M. caymanensis vs M. pallarii 1 3.74 2.16 0.17 0.115 M. caymanensis vs M. ripperi sp. nov. 1 5.65 2.17 0.03 0.114 M. pseudopallarii sp. nov. vs M. margoae sp. nov. 1 3.74 2.16 0.17 <0.001 variance with the first two components (32.8% for PC1 Integrative description of the topotypic and 27.0% for PC2). PERMANOVA showed an overall population of the species significant effect of the species on the PCs (p<0.001, Animals (measurements and statistics in Table 6): In Table 5). All of the post hoc pairwise PERMANOVA live animals, body almost transparent in smaller speci- comparisons, except some concerning M. caymanensis mens and whitish in larger animals; transparent after fix- (probably due to the very low sample size of this spe- ation in Hoyer’s medium (Fig. 3). Eyes present in live cies), were significant (Table 5). However, similar to ani- animals and after fixation in Hoyer’s medium. Small mal traits, egg measurements also did not separate the round and oval cuticular pores (0.5–1.5 μm in diameter) analyzed species (Fig. 2b). visible under both LCM and SEM scattered randomly throughout the entire body (Figs. 4a–e, 5a–d). Patches Taxonomic accounts of fine granulation on the external surface of legs I–III Phylum: Tardigrada Doyère, 1840 [76] as well as on the dorsal and dorsolateral sides of leg IV Class: Eutardigrada Richters, 1926 [77] visible in LCM (Fig. 4c, e) and SEM (Fig. 5b, d). A pulvi- Order: Parachela Schuster et al., 1980 [78] (restored by nus is present on the internal surface of legs I–III (Figs. [15]) 4d, 5c). In addition to the typical patches of leg granula- Superfamily: Macrobiotoidea Thulin, 1928 [79] (in [80]) tion, a band of granulation is present on the dorso- and Family: Macrobiotidae Thulin, 1928 [79] latero-caudal surface of the last body segment (Figs. 2, Genus: Macrobiotus C.A.S. Schultze, 1834 [18] 4a, 5a). It consists of two lateral patches of dense granu- Macrobiotus pallarii Maucci, 1954 [22] lation joined by a band of sparse dorsal granulation Material examined: 17 animals and 15 eggs. Specimens (Figs. 2, 4a, 5a). The sparse granulation also extends pos- were mounted on microscope slides in Hoyer’s medium teriorly from these dense granulation patches towards (9 animals + 10 eggs), fixed on SEM stubs (5+5), and the granulation on leg IV, but they never connect (Figs. processed for DNA sequencing (3+0). 2, 4a, 5a). This granulation can be poorly visible under Locality: 39°18′34.5″N, 16°32′19.9″E; 1436 m asl: Sil- LCM when the cuticle is wrinkled (Fig. 4b). vana Mansio, Cosenza, Italy: moss on rock in sparse for- Claws slender, of the hufelandi type. Primary est; coll. 16 December 2019 by Francesco Squillace. branches with distinct accessory points, a long com- Specimen depositories: Fourteen animals (slides: mon tract, and an evident stalk connecting the claw IT.337.03–11; SEM stub: 19.19) and 15 eggs (slides: to the lunula (Fig. 6a–f). Lunulae on all legs smooth IT.337.01–02; SEM stub: 19.19) were deposited at the and only sometimes faintly crenulated on leg IV (Fig. Institute of Zoology and Biomedical Research, Jagiello- 6a–f). Dark areas under each claw on legs I–III were nian University, Gronostajowa 9, 30-387, Kraków, often visible in LCM (Fig. 6a). Paired muscle attach- Poland. ments and faintly visible cuticular bars above them Stec et al. Zoological Letters (2021) 7:9 Page 9 of 45

Table 6 Measurements [in μm] of selected morphological structures of individuals of Macrobiotus pallarii Maucci, 1954 from the topotypic population (IT.337) mounted in Hoyer’s medium (N–number of specimens/structures measured, RANGE refers to the smallest and the largest structures among all measured specimens; SD–standard deviation) CHARACTER N RANGE MEAN SD μm pt μm pt μm pt Body length 9 421 – 617 1005 – 1413 536 1169 77 136 Buccal tube Buccal tube length 8 41.7 – 48.1 – 44.9 – 2.6 – Stylet support insertion point 8 32.5 – 37.3 77.1 – 79.4 35.0 77.9 1.9 0.8 Buccal tube external width 8 5.9 – 7.2 13.9 – 16.6 6.8 15.2 0.5 1.0 Buccal tube internal width 8 3.8 – 5.1 9.1 – 11.8 4.7 10.4 0.4 0.9 Ventral lamina length 8 22.6 – 28.2 51.4 – 59.4 25.3 56.4 1.8 2.8 Placoid lengths Macroplacoid 1 8 12.6 – 16.1 28.5 – 34.6 14.3 31.9 1.3 2.1 Macroplacoid 2 8 7.9 – 10.5 18.5 – 21.9 9.0 20.1 0.8 1.1 Microplacoid 8 3.3 – 5.1 7.5 – 11.8 4.2 9.3 0.6 1.4 Macroplacoid row 8 21.8 – 27.0 52.0 – 60.8 25.0 55.7 1.8 2.8 Placoid row 8 26.6 – 32.8 61.3 – 75.6 30.2 67.4 2.1 4.2 Claw 1 heights External primary branch 8 9.9 – 12.7 23.6 – 27.7 11.3 24.9 1.1 1.5 External secondary branch 7 7.9 – 10.0 18.2 – 23.1 8.6 19.3 0.8 1.7 Internal primary branch 8 8.4 – 12.7 20.1 – 25.4 10.5 23.0 1.4 1.8 Internal secondary branch 6 7.1 – 8.6 16.6 – 19.9 7.7 17.6 0.6 1.2 Claw 2 heights External primary branch 8 10.1 – 13.8 23.5 – 30.3 11.5 25.2 1.4 2.3 External secondary branch 8 8.4 – 10.3 18.1 – 23.8 9.1 20.3 0.8 1.8 Internal primary branch 8 9.3 – 12.4 21.7 – 27.7 10.7 23.4 1.3 2.2 Internal secondary branch 8 7.4 – 9.8 17.0 – 22.6 8.3 18.6 0.8 1.9 Claw 3 heights External primary branch 8 9.9 – 13.5 22.5 – 30.9 11.7 25.8 1.5 2.8 External secondary branch 8 7.7 – 10.4 17.7 – 23.3 9.1 20.1 1.1 2.0 Internal primary branch 8 8.7 – 12.6 20.9 – 29.1 10.9 24.1 1.5 2.7 Internal secondary branch 8 7.5 – 9.8 17.3 – 22.6 8.3 18.6 0.9 1.9 Claw 4 heights Anterior primary branch 7 11.7 – 14.3 26.6 – 30.6 13.0 28.6 0.9 1.7 Anterior secondary branch 6 8.8 – 10.2 19.3 – 22.2 9.5 21.1 0.5 1.1 Posterior primary branch 6 12.5 – 16.2 29.5 – 34.6 14.3 31.5 1.4 2.0 Posterior secondary branch 3 9.9 – 10.5 22.1 – 24.2 10.3 23.0 0.3 1.1 on legs I–III were often visible both with LCM and The first band of teeth is composed of numerous small SEM (Fig. 6a, d), whereas the horseshoe-shaped struc- teeth visible under LCM as granules (Fig. 7b–c) and ture connecting anterior and posterior claw IV was under SEM as cones (Fig. 8a–b), arranged in several visible only in LCM (Fig. 6b–c). rows, situated anteriorly in the oral cavity, just behind Mouth antero-ventral. Buccal apparatus of the Macro- the bases of the peribuccal lamellae. The second band of biotus type (Fig. 7a), with the ventral lamina and ten teeth is situated between the ring fold and the third peribuccal lamellae (Fig. 8a–b). The oral cavity armature band of teeth and comprises 3–4 rows of teeth visible was well developed and composed of three bands of with LCM as granules (Fig. 7b–c), and as cones in SEM teeth, all always clearly visible under LCM (Fig. 7b–c). (Fig. 8a–b) but larger than those in the first band. The Stec et al. Zoological Letters (2021) 7:9 Page 10 of 45

Fig. 3 Macrobiotus pallarii Maucci, 1954 from the topotypic population (IT.337) – habitus, adult specimen in dorso-ventral projection. Scale bar in μm most anterior row of teeth within the second band com- macroplacoids (2<1) and a microplacoid positioned close prises larger teeth than the subsequent posterior rows to them (i.e., the distance between the second macropla- (Fig. 7b–c). The teeth of the third band are located coid and the microplacoid is shorter than the micropla- within the posterior portion of the oral cavity, between coid length; Fig. 7a, d). The first macroplacoid is the second band of teeth and the buccal tube opening anteriorly narrowed and constricted in the middle, (Figs. 7b–c, 8a–b). The third band of teeth is divided whereas the second has a subterminal constriction (Fig. into the dorsal and ventral portions. Under both LCM 7d–e). and SEM, the dorsal teeth are seen as three distinct Eggs (measurements and statistics in Table 7): Laid transverse ridges, whereas the ventral teeth appear as freely, white, spherical with conical processes sur- two separate lateral transverse ridges, between which rounded by one row of areolae (Figs. 9, 10a–f). In SEM, one large tooth (sometimes circular in LCM) is visible multiple rings of tight annulation were visible on the en- (Figs. 7b–c, 8a–b). In SEM, teeth of the third band have tire process (Fig. 10a–c), although in some processes, faintly indented margins (Fig. 8a–b). Pharyngeal bulb annulation was present only in the upper portion of the spherical, with triangular apophyses, two rod-shaped process (Fig. 10d–f) (annulation not visible in LCM

Fig. 4 Macrobiotus pallarii Maucci, 1954 from the topotypic population (IT.337) – body and leg cuticle morphology seen with LCM: a–b – band of caudal granulation on the last body segment clearly visible in specimen with stretched cuticle (A) and hardly visible in specimen with wrinkled cuticle (b); c – granulation on the external surface of leg III; d – internal surface of leg III with evident pulvinus; e – granulation on dorsal surface of leg IV. Filled flat arrowheads indicate dense patches of granulation in the caudal band, filled indented arrowheads indicate sparse granulation in the caudal band, arrow indicates lateral gibbosity on the IV leg – a male secondary sexual character. Scale bar in μm Stec et al. Zoological Letters (2021) 7:9 Page 11 of 45

Fig. 5 Macrobiotus pallarii Maucci, 1954 from the topotypic population (IT.337) – body and leg cuticle morphology seen with SEM: a – band of caudal granulation on the last body segment; b – granulation on the external surface of leg II; c – internal surface of leg II with evident pulvinus; d – granulation on dorsal surface of leg IV. Filled flat arrowheads indicate dense patches of granulation in the caudal band, filled indented arrowheads indicate sparse granulation in the caudal band, arrow indicates lateral gibbosity – a male secondary sexual character. Scale bar in μm because it was obscured by the eminent labyrinthine visible as reticulation with circular meshes throughout layer). The upper parts of the processes are covered by the entire process (Fig. 9a–d). Small areas without re- granulation visible only under SEM, which to a varying ticulation are rarely present in some processes (Fig. 9b– extent is distributed on annuli (Fig. 10c–f). The labyrin- d). The upper part of the process is often elongated into thine layer between the process walls is present and short flexible apices (Figs. 9f–h, 10a–c), which are

Fig. 6 Macrobiotus pallarii Maucci, 1954 from the topotypic population (IT.337) – claw morphology: a–b – claws I and IV seen with LCM; c – magnification on lunulae IV seen with LCM; d–e – claws II and IV seen in SEM; f – magnification on lunulae IV seen with SEM. Empty indented arrowheads indicate dark circular areas under lunulae on the first three pairs of legs, filled flat arrowheads indicate cuticular bars above muscle attachments, empty flat arrowheads indicate double muscle attachments under claws, filled indented arrowheads indicate faintly visible indentations on lunulae IV, arrows indicate horseshoe structures connecting the anterior and posterior claws. Scale bars in μm Stec et al. Zoological Letters (2021) 7:9 Page 12 of 45

Fig. 7 Macrobiotus pallarii Maucci, 1954 from the topotypic population (IT.337) – buccal apparatus seen with LCM: a – an entire buccal apparatus; b–c – the oral cavity armature, dorsal and ventral teeth, respectively; d–e – placoid morphology, dorsal and ventral placoids, respectively. Filled flat arrowheads indicate the first band of teeth, empty flat arrowheads indicate the second band of teeth, filled indented arrowheads indicate the third band of teeth and empty indented arrowheads indicate central and subterminal constrictions in the first and second macroplacoid. Scale bars in μm occasionally absent or broken (Figs. 9e, 10d–f). The base flat (Fig. 10a–d), with the labyrinthine layer inside the of the processes extends into the six (only sometimes rims visible as bubbles in LCM (Fig. 9a–d). Areolae rims five) arms that form areolae rims (Figs. 9a–d, 10a–c). also delimit the areolae at the bases of processes, which Each process is surrounded by six (only sometimes five) forms an irregular collar around process bases (Figs. 9a– hexagonal areolae (Figs. 9a–d, 10a–c), which are occa- d, 10a–d) and makes the process bases penta- or hex- sionally falsely subdivided in the middle into two areolae agonal in the top view (Figs. 9a–d, 10a–b). The areola by a thin thickening perpendicular to the process base surface has wrinkles that are faintly visible under LCM (Figs. 9a–d, 10b). Areolae rims (walls) thick and usually (Fig. 9a–d) but clearly visible under SEM (Fig. 10a–d).

Fig. 8 Macrobiotus pallarii Maucci, 1954 from the topotypic population (IT.337) – the oral cavity armature seen with SEM: a–b – the oral cavity armature of a single specimen seen with SEM from different angles showing dorsal and ventral portion, respectively. Filled flat arrowheads indicate the first band of teeth, empty flat arrowheads indicate the second band of teeth, and filled indented arrowheads indicate the third band of teeth. Scale bars in μm Stec et al. Zoological Letters (2021) 7:9 Page 13 of 45

Table 7 Measurements [in μm] of selected morphological structures of the eggs of Macrobiotus pallarii Maucci, 1954 from the topotypic population (IT.337) mounted in Hoyer’s medium (N–number of eggs/structures measured, RANGE refers to the smallest and the largest structures among all measured specimens; SD–standard deviation) CHARACTER N RANGE MEAN SD Egg bare diameter 10 71.3 – 81.1 76.1 4.2 Egg full diameter 10 97.6 – 108.0 102.4 4.3 Process height 30 12.2 – 15.2 13.5 0.8 Process base width 30 14.1 – 21.0 16.4 1.7 Process base/height ratio 30 103% – 146% 121% 12% Interprocess distance 30 4.5 – 8.1 6.1 0.7 Number of processes on the egg circumference 10 10 – 12 11.2 0.8

Micropores are present within the areolae, but they are form of clearly visible lateral gibbosities on hind legs in distributed only around the areolae rims and are usually males (Figs. 4a, 5a, 11b). absent in the central part of the areola (Fig. 10b–d).

Reproduction DNA sequences and intraspecific genetic distances The species is dioecious. Spermathecae in females as well as testes in males have been found to be filled with  18S rRNA: GenBank: MT809069–71; 987 bp long; 1 spermatozoa, clearly visible under PCM up to 24 hours haplotype was found. after mounting in Hoyer’s medium (Fig. 11a–b). The  28S rRNA: GenBank: MT809081–3; 716 bp long; 1 species exhibits secondary sexual dimorphism in the haplotype was found.

Fig. 9 Macrobiotus pallarii Maucci, 1954 from the topotypic population (IT.337) – eggs seen with LCM: a–d – surface under ×1000 magnification of four different eggs; e–h – midsections of four different egg processes. Filled flat arrowheads indicate thickening perpendicular to the process base that divides the areola in the middle, filled indented arrowheads indicate areas of the egg processes without a reticulation/labyrinthine layer, and empty flat arrowheads indicate irregular collar around process bases. Scale bars in μm Stec et al. Zoological Letters (2021) 7:9 Page 14 of 45

Fig. 10 Macrobiotus pallarii Maucci, 1954 from the topotypic population (IT.337) – eggs seen with SEM: a – entire view of the egg; b–f – details of the egg surface between processes, areolation and egg processes. Filled flat arrowheads indicate thickening perpendicular to the process base that divides the areola in the middle, empty flat arrowheads indicate irregular collar around process bases. Scale bars in μm

 ITS-2: GenBank: MT809094–6; 362 bp long; 1 legs III and IV not extending posteriorly to the haplotype was found. granulation on leg IV (with sparse granulation ex-  COI: GenBank: MT807924–6; 630 bp long. 1 tending posteriorly to the granulation on leg IV in haplotype was found. M. pseudopallarii sp. nov.; see Fig. 1).  Macrobiotus ripperi sp. nov.: by faintly crenulated Phenotypic differential diagnosis lunulae IV (lunulae are dentate in M. ripperi sp. By having the processes surrounded by 5–6 areolae, it nov.), the presence of two lateral patches of dense resembles four other species of the Macrobiotus pallarii granulation between legs III and IV (patches of complex out of which three are newly described in this dense granulation are absent in M. ripperi sp. nov.; study. By the morphology of the animals and eggs, this see Fig. 1), the sparse granulation on the dorso- species can be differentiated specifically from the caudal end of the body connecting the dense granu- following: lation patches between legs III and IV not extending posteriorly to the granulation on legs IV (with the  Macrobiotus pseudopallarii sp. nov.: by faintly dense granulation patches between legs III and IV crenulated lunulae IV (lunulae are gently dentate in absent and the sparse granulation extending poster- M. pseudopallarii sp. nov.) and the sparse iorly to the granulation on legs IV in M. ripperi sp. granulation on the dorso-caudal end of the body nov.; see Fig. 1) and by the presence of granulation connecting the dense granulation patches between on the tips of egg processes (granulation is absent in Stec et al. Zoological Letters (2021) 7:9 Page 15 of 45

Fig. 11 Macrobiotus pallarii Maucci, 1954 from the topotypic population (IT.337) – reproduction (LCM): a – spermatheca (seminal vesicle) filled with spermatozoa and visible in females freshly mounted in Hoyer’s medium; b – testis filled with sperm visible in a male freshly mounted in Hoyer’s medium. The flat arrowhead indicates the female spermathecae, the indented arrowhead indicates the testis, and the arrow indicates the gibbosity on the IV leg. Scale bars in μm

M. ripperi sp. nov.; character visible only under not visible with LCM in M. caymanensis), the SEM). presence of granulation visible with LCM on all legs  Macrobiotus margoae sp. nov.:byfaintly (leg granulation is absent or not visible under LCM crenulated lunulae IV (lunulae are dentate in M. in M. caymanensis), a higher placoid row pt value margoae sp. nov.), the presence of two lateral patches (61.3–75.6 in M. pallarii vs. 47.8–59.9 in M. of dense granulation between legs III and IV (the caymanensis), and by the labyrinthine meshes within lateral patches of dense granulation are absent in M. the entire process wall (only small circular bubbles margoae sp. nov.; see Fig 1), the presence of sparse scattered randomly within the process wall are dorsal granulation between legs III and IV (this found in M. caymanensis). granulation absent in M. margoae sp. nov.;seeFig.1), all three bands of teeth in the oral cavity visible under Genotypic differential diagnosis LCM (the first band of teeth is not visible under LCM Interspecific genetic p-distances between M. pallarii and in M. margoae sp. nov.), a higher placoid row pt other species of the M. pallarii complex are as follows: value (61.3–75.6 in M. pallarii vs. 51.1–60.6 in M. margoae sp. nov.), the labyrinthine meshes within the  18S rRNA: 0.0–1.2% (0.7% on average), with the entire process wall (only small circular bubbles most similar being Macrobiotus pseudopallarii sp. scattered randomly within the process wall are found nov. from Montenegro (MT809065–7), and the in M. margoae sp. nov.), and by the presence of least similar being Macrobiotus margoae sp. nov. granulation on the tips of egg processes (granulation from the USA (MT809072–3). is absent in M. margoae sp. nov.; character visible  28S rRNA: 0.1–2.7% (1.8% on average), with the only under SEM). most similar being Macrobiotus pseudopallarii sp.  Macrobiotus caymanensis, known only from the nov. from Montenegro (MT809077–80), and the Cayman Islands: by all three bands of teeth in the least similar being Macrobiotus margoae sp. nov. oral cavity visible with LCM (the first band of teeth from the USA (MT809084–5). Stec et al. Zoological Letters (2021) 7:9 Page 16 of 45

 ITS-2: 0.8–6.7% (4.1% on average), with the most granulation on leg IV (Figs. 2, 13a, 14a–b). This granula- similar being Macrobiotus pseudopallarii sp. nov. tion is slightly visible under LCM when the cuticle is wrin- Haplotype 1 (H1) from Montenegro (MT809090–2), kled (Fig. 2, 13b). and the least similar being Macrobiotus margoae H2 Claws slender, of the hufelandi type. Primary branches sp. nov. from the USA (MT809097). with distinct accessory points, a long common tract, and an  COI: 14.0–21.1% (17.5% on average), with the most evident stalk connecting the claw to the lunula (Fig. 15a–f). similar being Macrobiotus pseudopallarii sp. nov. Lunulae on legs I–III smooth, whereas on leg IV gently den- Haplotype 2 (H2) from Montenegro (MT807920), tate (Fig. 15a–f). Dark areas under each claw on legs I–III and the least similar being Macrobiotus margoae sp. were faintly visible with LCM (Fig. 15a). Paired muscle at- nov. from USA (MT807927–8). tachments and faintly visible cuticular bars above them on legs I–III were often visible both with LCM (Fig. 15a) and Macrobiotus pseudopallarii Stec, Vecchi & SEM, whereas the horseshoe-shaped structure connecting Michalczyk, sp. nov. anterior and posterior claws IV was visible only under LCM Macrobiotus cf. pallarii ME.007 [19] (Fig. 15b–c). Zoobank: urn:lsid:zoobank.org:act:2F0C8594-A645- Mouth antero-ventral. Buccal apparatus of the Macro- 4146-86A2-99C7BF9C307C biotus type (Fig. 16a), with the ventral lamina and ten Etymology: The name refers to the morphology of the peribuccal lamellae (Fig. 17a–b). The oral cavity arma- new species, which highly resembles that of Macrobiotus ture was well developed and composed of three bands of pallarii (Latin “pseudo” = “false”). teeth, all always clearly visible under LCM (Fig. 16b–c). Material examined: 96 animals and 130 eggs. Speci- The first band of teeth is composed of numerous small mens were mounted on microscope slides in Hoyer’s teeth visible with LCM as granules (Fig. 16b–c) and with medium (76 animals + 116 eggs), fixed on SEM stubs SEM as cones (Fig. 17a–b), arranged in several rows, sit- (15+14), and processed for DNA sequencing (5+0). uated anteriorly in the oral cavity, just behind the bases Type locality: 42°47′57.54″N, 19°27′18.47″E; 1015 m of the peribuccal lamellae. The second band of teeth is asl: Montenegro: Crkvine; moss on stone; coll. May 2018 situated between the ring fold and the third band of by Aleksandra Rysiewska. teeth and comprises 3–4 rows of teeth visible with LCM Type depositories: Holotype (slide ME.007.05 with 9 as granules (Fig. 16b–c) and with SEM as cones (Fig. paratypes), 81 paratypes (slides: ME.007.06–10; SEM 17a–b) but larger than those in the first band. The most stub: 18.09) and 130 eggs (slides: ME.007.01–04; SEM anterior row of teeth within the second band comprises stub: 18.09) were deposited at the Institute of Zoology larger teeth than the subsequent posterior rows (Fig. and Biomedical Research, Jagiellonian University, Gro- 16b–c). The teeth of the third band are located within nostajowa 9, 30-387, Kraków, Poland. the posterior portion of the oral cavity, between the sec- ond band of teeth and the buccal tube opening (Figs. Description of the new species 16b–c, 17a–b). The third band of teeth is divided into Animals (measurements and statistics in Table 8): In live the dorsal and ventral portions. Under both LCM and animals, body almost transparent in smaller specimens SEM, the dorsal teeth are seen as three distinct trans- and whitish in larger animals; transparent after fixation in verse ridges, whereas the ventral teeth appear as two Hoyer’smedium(Fig.12). Eyes present in live animals and separate lateral transverse ridges, between which one after fixation in Hoyer’s medium. Small round and oval large tooth (sometimes circular in LCM) is visible (Figs. cuticular pores (0.5–1.2 μm in diameter), visible under 16b–c, 17a–b). In SEM, teeth of the third band have in- both LCM and SEM, scattered randomly throughout the dented margins (Fig. 17a–b). Pharyngeal bulb spherical, entire body (Figs. 13a–e, 14a–e). Patches of fine granula- with triangular apophyses, two rod-shaped macropla- tion on the external surface of legs I–III as well as on the coids (2<1) and a microplacoid positioned close to them dorsal and dorsolateral sides of leg IV visible with LCM (i.e., the distance between the second macroplacoid and (Fig. 13c, e) and SEM (Fig. 14c, e). A pulvinus is present the microplacoid is shorter than the microplacoid on the internal surface of legs I–III (Figs. 13d, 14d). In length; Fig. 16d–e). The first macroplacoid is anteriorly addition to the typical patches of leg granulation, a band narrowed and constricted in the middle, whereas the of granulation is present on the dorso- and latero-caudal second has a subterminal constriction (Fig. 16d–e). surface of the last body segment (Figs. 2, 13a, 14a–b). It Eggs (measurements and statistics in Table 9): Laid consists of two lateral patches of dense granulation, joined freely, white, spherical with conical processes sur- with each other by a band of sparse dorsal granulation rounded by one row of areolae (Figs. 18a–h, 19a–f). In (Figs. 2, 13a, 14a–b). The sparse granulation also extends SEM, multiple rings of tight annulation on the entire anteriorly and posteriorly from those dense granulation process surface were visible (Fig. 19a–b), although in patches with posterior extension, which connects with the some processes, annulation was present only in the Stec et al. Zoological Letters (2021) 7:9 Page 17 of 45

Table 8 Measurements [in μm] of selected morphological structures of individuals of Macrobiotus pseudopallarii sp. nov. from Montenegro (ME.007) mounted in Hoyer’s medium (N–number of specimens/structures measured, RANGE refers to the smallest and the largest structures among all measured specimens; SD–standard deviation) CHARACTER N RANGE MEAN SD Holotype μm pt μm pt μm pt μm pt Body length 30 386 – 580 957 – 1328 507 1215 45 78 534 1275 Buccal tube Buccal tube length 30 36.3 – 47.8 – 41.8 – 3.0 – 41.9 – Stylet support insertion point 30 27.9 – 37.9 76.1 – 79.8 32.5 77.8 2.5 1.1 32.3 77.1 Buccal tube external width 30 5.2 – 8.5 13.3 – 19.4 6.8 16.2 0.8 1.4 6.9 16.5 Buccal tube internal width 30 3.7 – 6.4 10.2 – 14.6 5.2 12.3 0.6 1.0 4.9 11.7 Ventral lamina length 30 21.6 – 29.7 58.3 – 64.8 26.1 62.4 2.0 1.9 25.2 60.1 Placoid lengths Macroplacoid 1 30 8.3 – 16.3 22.1 – 39.4 13.6 32.5 2.0 4.0 14.1 33.7 Macroplacoid 2 30 6.5 – 10.6 17.3 – 24.8 8.9 21.4 1.1 1.8 8.7 20.8 Microplacoid 30 3.3 – 5.6 7.1 – 12.8 4.1 9.9 0.5 1.3 4.3 10.3 Macroplacoid row 30 19.3 – 27.0 52.1 – 62.5 24.1 57.7 2.1 2.7 24.1 57.5 Placoid row 30 23.8 – 32.9 65.0 – 75.1 29.5 70.6 2.6 2.7 30.2 72.1 Claw 1 heights External primary branch 24 10.0 – 13.1 22.8 – 33.1 11.4 27.5 1.0 2.3 11.7 27.9 External secondary branch 24 7.6 – 12.5 18.2 – 28.1 9.1 21.8 1.2 2.7 10.1 24.1 Internal primary branch 26 9.5 – 12.4 21.9 – 31.2 11.0 26.4 0.8 2.3 11.6 27.7 Internal secondary branch 26 6.9 – 11.5 16.0 – 26.5 8.7 21.0 1.2 2.8 8.6 20.5 Claw 2 heights External primary branch 26 10.9 – 13.4 25.2 – 34.3 12.0 28.8 0.7 2.3 12.2 29.1 External secondary branch 26 7.8 – 11.8 18.5 – 29.8 9.5 22.9 1.1 2.8 10.2 24.3 Internal primary branch 28 9.9 – 12.7 24.0 – 33.9 11.2 26.9 0.7 2.2 11.6 27.7 Internal secondary branch 27 7.0 – 10.3 17.3 – 27.0 8.6 20.7 0.8 2.2 9.2 22.0 Claw 3 heights External primary branch 28 10.0 – 13.6 24.5 – 35.8 12.0 29.0 0.9 2.8 12.3 29.4 External secondary branch 26 7.1 – 11.3 18.5 – 26.5 9.4 22.6 1.0 2.4 10.1 24.1 Internal primary branch 26 9.5 – 12.9 22.2 – 32.9 11.3 27.0 0.9 2.3 11.5 27.4 Internal secondary branch 23 6.7 – 10.8 16.0 – 26.2 8.6 20.7 1.0 2.6 9.0 21.5 Claw 4 heights Anterior primary branch 16 11.6 – 13.8 28.2 – 36.0 13.0 31.2 0.6 2.1 12.4 29.6 Anterior secondary branch 16 7.8 – 10.5 19.1 – 26.4 9.3 22.4 0.7 2.2 10.0 23.9 Posterior primary branch 12 11.6 – 15.1 27.6 – 38.5 13.4 32.2 1.1 3.0 14.0 33.4 Posterior secondary branch 11 7.1 – 11.5 16.5 – 27.4 10.0 23.5 1.3 3.1 11.5 27.4 upper portion of the process (Fig. 19c–f) (annulation not are present in some processes (Fig. 18a, c); however, very visible with LCM because it was obscured by the emi- rarely, the reticulation can also be considerably reduced nent labyrinthine layer). The upper parts of the pro- (Fig. 18d). The upper part of the process is often elon- cesses are covered by granulation, which to a varying gated into short flexible apices (Figs. 18e–h, 19c–f), extent is distributed on annuli, visible only under SEM which can be occasionally broken. The bottom part of (Fig. 19c–f). The labyrinthine layer within the process the processes is flattened and extends into the six (only walls is present and visible as reticulation with circular/ sometimes five) arms that form areolae rims (Figs. 18a– ellipsoidal meshes throughout the entire process (Fig. d, 19a–d). Each process is surrounded by six (only some- 18a–c). Only sometimes small areas without reticulation times five) hexagonal areolae (Figs. 18a–d, 19a–d), which Stec et al. Zoological Letters (2021) 7:9 Page 18 of 45

Fig. 12 Macrobiotus pseudopallarii sp. nov. from Montenegro (ME.007) – habitus, adult specimen in dorso-ventral projection (holotype). Scale bar in μm are occasionally falsely subdivided in the middle into areolae, but they are distributed only around the two areolae by a thin thickening perpendicular to the areola rims and are usually absent in the central part process base (Figs. 18c, 19b–d). Areolae rims (walls) of the areola (Fig. 19b–d). thick and usually flat (Fig. 19a–d), with the labyrin- thine layer inside the rims visible as bubbles under LCM (Fig. 18a–d). Areolae rims also delimit the Reproduction areolae at the bases of processes, which forms an ir- The species is dioecious. Spermathecae in females as regular collar around process bases (Figs. 18a–d, well as testes in males were found to be filled with 19a–d) and makes the process bases penta- or hex- spermatozoa, clearly visible under PCM up to 24 hours agonal in the top view (Figs. 18a–d, 19a–b). The after mounting in Hoyer’s medium (Fig. 20a–b). The areola surface has wrinkles that are faintly visible species exhibits secondary sexual dimorphism in the under LCM (Fig. 18a–d) but clearly visible under form of clearly visible lateral gibbosities on hind legs in SEM (Fig. 19a–d). Micropores are present within the males (Fig. 20b).

Fig. 13 Macrobiotus pseudopallarii sp. nov. from Montenegro (ME.007) – body and leg cuticle morphology seen with LCM: a–b – band of caudal granulation on the last body segment clearly visible in specimen with stretched cuticle (a) and hardly visible in specimen with wrinkled cuticle (b); c – granulation on the external surface of leg III; d – internal surface of leg III with evident pulvinus; e – granulation on dorsal surface of leg IV. Filled flat arrowheads indicate dense patches of granulation in the caudal band, filled indented arrowheads indicate sparse granulation in the caudal band. Scale bar in μm Stec et al. Zoological Letters (2021) 7:9 Page 19 of 45

Fig. 14 Macrobiotus pseudopallarii sp. nov. from Montenegro (ME.007) – body and leg cuticle morphology seen with SEM: a–b – band of caudal granulation on the last body segment; c – granulation on the external surface of leg II; d – internal surface of leg II with evident pulvinus; e – granulation on dorsal surface of leg IV. Filled flat arrowheads indicate dense patches of granulation in the caudal band, filled indented arrowheads indicate sparse granulation in the caudal band. Scale bar in μm

Fig. 15 Macrobiotus pseudopallarii sp. nov. from Montenegro (ME.007) – claw morphology: a–b – claws III and IV seen with LCM; C – magnification of lunulae IV seen with LCM; d–e – claws III and IV seen with SEM; f – magnification of lunulae IV seen with SEM. Empty-indented arrowheads indicate dark circular areas under lunulae on the first three pairs of legs, filled flat arrowheads indicate cuticular bars above muscle attachments, and arrows indicate horseshoe structures connecting the anterior and posterior claws. Scale bars in μm Stec et al. Zoological Letters (2021) 7:9 Page 20 of 45

Fig. 16 Macrobiotus pseudopallarii sp. nov. from Montenegro (ME.007) – buccal apparatus seen with LCM: a – an entire buccal apparatus; b–c – the oral cavity armature, dorsal and ventral teeth, respectively; d–e – placoid morphology, dorsal and ventral placoids, respectively. Filled flat arrowheads indicate the first band of teeth, empty flat arrowheads indicate the second band of teeth, filled indented arrowheads indicate the third band of teeth and empty indented arrowheads indicate central and subterminal constrictions in the first and second macroplacoid. Scale bars in μm

DNA sequences and intraspecific genetic distances  ITS-2 sequences (GenBank: MT809090–2), 362 bp long; 2 haplotypes were found, separated by a p-  18S rRNA sequences (GenBank: MT809065–8), distance of 0.6%. 798–897bplong;1haplotypewasfound.  COI sequences (GenBank: MT807920–2), 630 bp  28S rRNA sequences (GenBank: MT809077–80), long; 2 haplotypes were found, separated by a p- 690–716 bp long; 1 haplotype was found. distance of 0.3%.

Fig. 17 Macrobiotus pseudopallarii sp. nov. from Montenegro (ME.007) – the oral cavity armature seen with SEM: a–b – the oral cavity armature of a single specimen seen with SEM from different angles showing dorsal and ventral portion, respectively. Filled flat arrowheads indicate the first band of teeth, empty flat arrowheads indicate the second band of teeth, and filled indented arrowheads indicate the third band of teeth. Scale bars in μm Stec et al. Zoological Letters (2021) 7:9 Page 21 of 45

Table 9 Measurements [in μm] of selected morphological structures of the eggs of Macrobiotus pseudopallarii sp. nov. from Montenegro (ME.007) mounted in Hoyer’s medium (N–number of eggs/structures measured, RANGE refers to the smallest and the largest structures among all measured specimens; SD–standard deviation) CHARACTER N RANGE MEAN SD Egg bare diameter 30 63.8 – 81.8 73.8 4.3 Egg full diameter 30 85.7 – 115.4 103.0 7.4 Process height 90 12.1 – 23.4 16.2 2.4 Process base width 90 10.2 – 21.0 15.5 1.9 Process base/height ratio 90 67% – 139% 97% 16% Interprocess distance 90 2.0 – 8.4 4.5 1.5 Number of processes on the egg circumference 30 10 – 14 12.5 1.0

Phenotypic differential diagnosis patches between legs III and IV extending By having the processes surrounded by 5–6 areolae, it posteriorly to the granulation on legs IV (sparse resembles four other species of the Macrobiotus pallarii granulation does not extend posteriorly to the complex out of which two are newly described in this granulation on legs IV in M. pallarii; see Fig. 1). study. By the morphology of the animals and eggs, this  Macrobiotus ripperi sp. nov.: by gently dentate species can be differentiated from the following: lunulae IV (lunulae are clearly dentate in M. ripperi sp. nov.), by the presence of two lateral patches of  Macrobiotus pallarii: by gently dentate lunulae IV dense granulation between legs III and IV (dense (lunulae are faintly crenulated in M. pallarii) and a granulation patches between legs III and IV are sparse granulation connecting the dense granulation absent in M. ripperi sp. nov.; see Fig. 1) and by the

Fig. 18 Macrobiotus pseudopallarii sp. nov. from Montenegro (ME.007) – eggs seen with LCM: a–d – surface under ×1000 magnification of four different eggs; e–h – midsections of four different egg processes. The filled flat arrowhead indicates thickening perpendicular to the process base that divides the areola in the middle, filled indented arrowheads indicate areas of the egg processes without a reticulation/labyrinthine layer, and empty flat arrowheads indicate irregular collars around process bases. Scale bars in μm Stec et al. Zoological Letters (2021) 7:9 Page 22 of 45

Fig. 19 Macrobiotus pseudopallarii sp. nov. from Montenegro (ME.007) – eggs seen with SEM: a – entire view of the egg; b–f – details of the egg surface between processes, areolation and egg processes. Filled flat arrowheads indicate thickening perpendicular to the process base that divides the areola in the middle, empty flat arrowheads indicate irregular collars around process bases. Scale bars in μm

presence of granulation on the egg process tips nov. vs. 51.1–60.6 in M. margoae sp. nov.), by the (granulation is absent in M. ripperi sp. nov.; presence of meshes within the entire process walls character visible only under SEM). (only small circular bubbles scattered randomly  Macrobiotus margoae sp. nov.: by having an oral within the process are found in M. margoae sp. cavity armature well developed and composed of nov.), and by the presence of granulation on the egg three bands of teeth visible under LCM (the oral process tips (granulation is absent in M. margoae sp. cavity armature is less developed, and the first band nov.; character visible only under SEM). of teeth are not visible under LCM in M. margoae  Macrobiotus caymanensis: by having an oral cavity sp. nov.), by having lunulae IV gently dentate armature well developed and composed of three (lunulae are clearly dentate in M. margoae sp. nov.), bands of teeth visible under LCM (the oral cavity by the presence of two lateral patches of dense armature is less developed, and the first band of granulation between legs III and IV (dense teeth is not visible under LCM in M. caymanensis), granulation patches between legs III and IV are by the presence of granulation visible with LCM in absent in M. margoae sp. nov.; see Fig. 1), by the all legs (granulation is not visible with LCM in M. presence of sparse dorsal granulation between legs caymanensis), by having lunulae IV gently dentate III and IV (sparse granulation is absent in M. (the lunulae are smooth in M. caymanensis), by a margoae sp. nov.; see Fig. 1), by a higher placoid higher placoid row pt value (65.6–75.1 in M. row pt value (65.0–75.1 in M. pseudopallarii sp. pseudopallarii sp. nov. vs. 47.8–59.9 in M. Stec et al. Zoological Letters (2021) 7:9 Page 23 of 45

Fig. 20 Macrobiotus pseudopallarii sp. nov. from Montenegro (ME.007) – reproduction (LCM): a – spermatheca (seminal vesicle) filled with spermatozoa and visible in females freshly mounted in Hoyer’s medium; b – testis filled with sperm visible in a male freshly mounted in Hoyer’s medium. The flat arrowhead indicates the female spermathecae, the indented arrowhead indicates the testis, and the arrow indicates the gibbosity on the IV leg. Scale bars in μm

caymanensis) and by the presence of meshes within  ITS-2: 0.8–6.9% (5.1% on average), with the most the entire process (only small circular bubbles similar being Macrobiotus pallarii from Italy scattered randomly within the process walls are (MT809094–6) and the least similar being found in M. caymanensis). Macrobiotus margoae sp. nov. H2 from USA (MT809097).  COI: 14.0–22.5% (19.9% on average), with the most Genotypic differential diagnosis similar being Macrobiotus pallarii from Italy Interspecific genetic p-distances between M. pseudopal- (MT807924–6) and the least similar being larii sp. nov. and other species of the M. pallarii com- Macrobiotus ripperi sp. nov. Haplotype 3 (H3) from plex are as follows: Finland (MT807930–2).

 18S rRNA: 0.0–1.4% (0.7% on average), with the Macrobiotus ripperi Stec, Vecchi & Michalczyk, sp. most similar being Macrobiotus pallarii from Italy nov. (MT809069–71) and the least similar being Macrobiotus pallarii in [81] Macrobiotus margoae sp. nov. from the USA Macrobiotus cf. pallarii in [82] (MT809072–3). Macrobiotus cf. pallarii PL.015 [19]  28S rRNA: 0.1–2.6% (1.7% on average), with the Macrobiotus cf. pallarii FI.066 [19] most similar being Macrobiotus pallarii from Italy Zoobank: urn:lsid:zoobank.org:act:EBA3D361-F598- (MT809081–3) and the least similar being 4679-8503-11862D10240D Macrobiotus margoae sp. nov. from the USA Etymology: Named after “Ripper”, the giant (MT809084–5). tardigrade-like creature from the TV series “Star Trek: Stec et al. Zoological Letters (2021) 7:9 Page 24 of 45

Table 10 Measurements [in μm] of selected morphological structures of individuals of Macrobiotus ripperi sp. nov. from Poland (PL.015) mounted in Hoyer’s medium (N–number of specimens/structures measured, RANGE refers to the smallest and the largest structures among all measured specimens; SD–standard deviation) CHARACTER N RANGE MEAN SD Holotype μm pt μm pt μm pt μm pt Body length 30 384 – 491 936 – 1233 447 1133 28 69 444 1223 Buccal tube Buccal tube length 30 35.4 – 43.4 – 39.5 – 1.7 – 36.3 – Stylet support insertion point 30 28.0 – 33.1 75.3 – 79.1 30.2 76.6 1.3 0.9 28.1 77.4 Buccal tube external width 30 5.1 – 6.6 13.3 – 16.4 6.0 15.1 0.4 0.8 5.7 15.7 Buccal tube internal width 30 3.9 – 5.0 10.0 – 12.7 4.5 11.5 0.3 0.8 4.2 11.6 Ventral lamina length 30 20.6 – 26.3 54.1 – 63.6 23.2 58.7 1.5 2.5 20.6 56.7 Placoid lengths Macroplacoid 1 30 10.0 – 14.4 26.6 – 34.8 12.3 31.2 1.0 2.2 11.5 31.7 Macroplacoid 2 30 6.8 – 9.7 17.5 – 23.3 7.8 19.9 0.7 1.6 7.1 19.6 Microplacoid 30 2.4 – 4.9 6.5 – 12.4 3.7 9.4 0.6 1.3 3.5 9.6 Macroplacoid row 30 19.4 – 25.3 50.1 – 61.1 21.7 55.1 1.3 2.5 20.3 55.9 Placoid row 30 24.0 – 29.1 62.1 – 71.9 26.8 67.9 1.5 2.8 24.7 68.0 Claw 1 heights External primary branch 29 9.4 – 12.5 24.4 – 30.6 11.1 28.2 0.8 1.8 11.1 30.6 External secondary branch 29 6.2 – 9.8 16.0 – 24.1 7.9 20.1 1.0 2.3 7.8 21.5 Internal primary branch 28 8.7 – 11.9 22.7 – 30.5 10.4 26.5 0.7 1.7 9.9 27.3 Internal secondary branch 28 6.3 – 9.0 16.1 – 22.6 7.5 19.2 0.5 1.5 7.5 20.7 Claw 2 heights External primary branch 29 10.0 – 13.3 25.1 – 32.8 11.7 29.8 0.8 2.0 11.3 31.1 External secondary branch 28 6.8 – 10.1 17.5 – 26.4 8.4 21.4 0.9 2.2 8.2 22.6 Internal primary branch 29 7.7 – 13.6 19.9 – 34.1 10.7 27.2 1.0 2.4 10.6 29.2 Internal secondary branch 30 6.2 – 9.7 16.3 – 24.3 7.9 19.9 0.9 1.9 7.8 21.5 Claw 3 heights External primary branch 30 10.0 – 13.0 24.2 – 33.4 11.7 29.6 0.9 2.2 11.4 31.4 External secondary branch 29 7.0 – 10.0 17.5 – 25.2 8.7 21.9 0.8 2.0 8.4 23.1 Internal primary branch 30 9.3 – 12.6 24.2 – 32.2 10.8 27.5 0.7 1.9 10.7 29.5 Internal secondary branch 30 5.8 – 9.6 15.1 – 24.3 7.9 19.9 1.0 2.2 7.9 21.8 Claw 4 heights Anterior primary branch 30 11.2 – 14.8 28.2 – 36.1 12.7 32.3 0.9 2.4 13.1 36.1 Anterior secondary branch 27 6.9 – 10.9 18.2 – 26.9 9.0 22.8 1.0 2.4 9.4 25.9 Posterior primary branch 28 12.4 – 15.8 30.9 – 41.0 14.0 35.5 1.0 2.8 14.9 41.0 Posterior secondary branch 13 7.0 – 10.4 17.9 – 25.7 8.9 22.8 1.0 2.6 ? ?

Discovery” to celebrate the presence of tardigrades in Type depositories: Holotype (slide PL.015.06 with 14 pop culture. paratypes), 183 paratypes (slides: PL.015.07–14; SEM Material examined: 202 animals and 77 eggs. Speci- stub: 18.06) and 77 eggs (slides: PL.015.01–05; SEM mens were mounted on microscope slides in Hoyer’s stub: 18.06) were deposited at the Institute of Zoology medium (178 animals + 62 eggs), fixed on SEM stubs and Biomedical Research, Jagiellonian University, Gro- (20+15), and processed for DNA sequencing (4+0). nostajowa 9, 30-387, Kraków, Poland. Type locality: 49°42′09″N, 21°55′53″E; 389 m asl: Additional material: Poland: Malinówka, Yew Reserve; moss from forest; coll. 46 animals and 52 eggs. Specimens were mounted on April 2014 by Piotr Gąsiorek. microscope slides in Hoyer’s medium (42 animals + 52 Stec et al. Zoological Letters (2021) 7:9 Page 25 of 45

Fig. 21 Macrobiotus ripperi sp. nov. from Poland (PL.015) – habitus, adult specimen in dorso-ventral projection (holotype). Scale bar in μm eggs) and processed for DNA sequencing (4+0). Locality: of fine granulation on the external surface of legs I–III 62°13′24.6″N, 25°46′20.4″E; 84 m asl: Finland: Jyväs- as well as on the dorsal and dorsolateral sides of leg IV kylä, Graniitti, moss on rock at the entrance to a bunker; visible with LCM (Fig. 22c, e) and SEM (Fig. 23d, f). A coll. 8th Feb 2019 by Matteo Vecchi. Specimen depositor- pulvinus is present on the internal surface of legs I–III ies: Forty-two animals (slides: FI.066.05–08) and 52 eggs (Figs. 22d, 23e). In addition to the typical patches of leg (slides: FI.066.01–04) were deposited at the Institute of granulation, sparse and uniformly distributed granula- Zoology and Biomedical Research, Jagiellonian University, tion is also present on the dorso- and latero-caudal sur- Gronostajowa 9, 30-387, Kraków, Poland. face of the last body segment (Figs. 22a, 23a–c). The sparse granulation connects with denser granulation Description of the new species patches on leg IV (Figs. 2, 22a, 23a–c). This granulation Animals (measurements and statistics in Table 10): In is slightly visible with LCM when the cuticle is wrinkled live animals, body almost transparent in smaller speci- (Fig. 22b). mens and whitish in larger animals; transparent after fix- Claws slender, of the hufelandi type. Primary branches ation in Hoyer’s medium (Fig. 21). Eyes present in live with distinct accessory points, a long common tract, and an animals and after fixation in Hoyer’s medium. Small evident stalk connecting the claw to the lunula (Fig. 24a–f). round and oval cuticular pores (0.5–1.4 μm in diameter), Lunulae on legs I–III smooth, whereas on legs IV usually visible under both LCM and SEM, scattered randomly clearly dentate (Fig. 24a, c–f). Dentation was rarely absent or throughout the entire body (Figs. 22a–e, 23a–f). Patches most likely not visible under LCM (Fig. 24b). Dark areas

Fig. 22 Macrobiotus ripperi sp. nov. from Poland (PL.015) – body and leg cuticle morphology seen with LCM: a–b – band of caudal granulation on the last body segment clearly visible in specimen with stretched cuticle (a) and hardly visible in specimen with wrinkled cuticle (b); c – granulation on the external surface of leg III; d – internal surface of leg II with evident pulvinus; e – granulation on dorsal surface of leg IV. Filled indented arrowheads indicate sparse granulation in the caudal band. Scale bar in μm Stec et al. Zoological Letters (2021) 7:9 Page 26 of 45

Fig. 23 Macrobiotus ripperi sp. nov. from Poland (PL.015) – body and leg cuticle morphology seen with SEM: a–c – band of caudal granulation on the last body segment; d – granulation on the external surface of leg II; e – internal surface of leg III with evident pulvinus; f – granulation on dorsal surface of leg IV. Filled indented arrowheads indicate sparse granulation in the caudal band, arrows indicate lateral gibbosities on the IV leg – a male secondary sexual character. Scale bar in μm under each claw on legs I–IIIwerefaintlyvisibleunderLCM band of teeth and comprises 3–4 rows of teeth visible (Fig. 24a). Paired muscle attachments and faintly visible cu- under LCM as granules (Fig. 25b–c) and under SEM as ticular bars above them on legs I–IIIwereoftenvisiblewith cones (Fig. 26a–b) but larger than those in the first both LCM (Fig. 24a) and SEM (Fig. 24d), whereas the band. The most anterior row of teeth within the second horseshoe-shaped structure connecting anterior and poster- band comprises larger teeth than the subsequent poster- ior claw IV was visible only with LCM (Fig. 24b–c). ior rows (Fig. 25b–c). The teeth of the third band are lo- Mouth antero-ventral. Buccal apparatus of the Macro- cated within the posterior portion of the oral cavity, biotus type (Fig. 25a), with the ventral lamina and ten between the second band of teeth and the buccal tube peribuccal lamellae (Fig. 26a–b). The oral cavity arma- opening (Figs. 25b–c, 26a–b). The third band of teeth is ture was well developed and composed of three bands of divided into the dorsal and ventral portions. Under both teeth, all always clearly visible under LCM (Fig. 25b–c). LCM and SEM, the dorsal teeth are seen as three dis- The first band of teeth is composed of numerous small tinct transverse ridges, whereas the ventral teeth appear teeth visible under LCM as granules (Fig. 25b–c) and as two separate lateral transverse ridges, between which under SEM as cones (Fig. 26a–b), arranged in several one large tooth (sometimes circular in LCM) is visible rows, situated anteriorly in the oral cavity, just behind (Figs. 25b–c, 26a–b). In SEM, teeth of the third band the bases of the peribuccal lamellae. The second band of have indented margins (Fig. 26a–b). Pharyngeal bulb teeth is situated between the ring fold and the third spherical, with triangular apophyses, two rod-shaped Stec et al. Zoological Letters (2021) 7:9 Page 27 of 45

Fig. 24 Macrobiotus ripperi sp. nov. from Poland (PL.015) – claw morphology: a–b – claws II and IV seen with LCM; c – magnification of lunulae IV seen with LCM; d–e – claws I and IV seen with SEM; f – magnification of lunulae IV seen with SEM. Empty indented arrowhead indicates dark circular areas under lunulae on the first three pairs of legs, filled flat arrowheads indicate cuticular bar above muscle attachments, empty flat arrowheads indicate double muscle attachments under claws, arrows indicate horseshoe structure connecting the anterior and the posterior claw. Scale bars in μm

Fig. 25 Macrobiotus ripperi sp. nov. from Poland (PL.015) – buccal apparatus seen with LCM: a – an entire buccal apparatus; b–c – the oral cavity armature, dorsal and ventral teeth, respectively; d–e – placoid morphology, dorsal and ventral placoids, respectively. Filled flat arrowheads indicate the first band of teeth, empty flat arrowheads indicate the second band of teeth, filled indented arrowheads indicate the third band of teeth, and empty indented arrowheads indicate central and subterminal constrictions in the first and second macroplacoids. Scale bars in μm Stec et al. Zoological Letters (2021) 7:9 Page 28 of 45

Fig. 26 Macrobiotus ripperi sp. nov. from Poland (PL.015) – the oral cavity armature seen with SEM: a–b – the oral cavity armature of a single specimen seen with SEM from different angles showing dorsal and ventral portion, respectively. Filled flat arrowheads indicate the first band of teeth, empty flat arrowheads indicate the second band of teeth, and filled indented arrowheads indicate the third band of teeth. Scale bars in μm macroplacoids (2<1) and a microplacoid positioned close (Fig. 27d). The upper part of the process surface is often to them (i.e., the distance between the second macropla- elongated into short flexible apices that can be occasion- coid and the microplacoid is shorter than the micropla- ally broken and that can sometimes be bifurcated and coid length; Fig. 25d–e). The first macroplacoid is have bubble-like structures (Figs. 27e–h, 28a, e–f). The anteriorly narrowed and constricted in the middle, base of the processes extends into the six (only sometimes whereas the second has a subterminal constriction (Fig. five) arms that form areolae rims (Figs. 27a–d, 28a–f). 25d–e). Each process is surrounded by six (only sometimes five) Eggs (measurements and statistics in Table 11): Laid hexagonal areolae (Figs. 27a–d, 28a–d), which are occa- freely, white, spherical with conical processes surrounded sionally falsely subdivided in the middle into two areolae by one row of areolae (Figs. 27a–h, 28a–f). In SEM, mul- by a thin thickening perpendicular to the process base tiple rings of tight annulation on the entire process surface (Figs. 27a–c, 28a, c). Areolae rims (walls) are usually thick were visible (Fig. 28a–b), although in some processes, an- and flat (Fig. 28a, c–d), but sometimes they can also be nulation was present only in the upper portion of the very thin (Fig. 28b), with the labyrinthine layer inside the process (Fig. 28e–f) (annulation not visible in LCM be- rims visible as bubbles under LCM (Fig. 27a–d). The cause it was obscured by the eminent labyrinthine layer). areola surface has wrinkles that are faintly visible under The upper parts of the processes are smooth and not cov- LCM (Fig. 27a–d) but clearly visible under SEM (Fig. ered with granulation (Fig. 28e–f). The labyrinthine layer 28a–f). Micropores are present within the areolae, but between the process walls was present and usually visible they are distributed only around the areolae rims and usu- only in the bottom part of egg processes as one to three ally absent in the central part of the areola (Fig. 28b–f). rows of meshes and some bubbles scattered randomly on the remaining process (Fig. 27a–c). These rings of reticu- lation can also sometimes be visible even with SEM (Fig. Reproduction 28c, e). Sometimes well-developed reticulation with circu- The species is dioecious. Spermathecae in females as lar/ellipsoidal meshes is observed in the entire process but well as testes in males were found to be filled with still with clearly visible places deprived of the reticulation spermatozoa, clearly visible under PCM up to 24 hours after mounting in Hoyer’s medium (Fig. 29a–b). The

Table 11 Measurements [in μm] of selected morphological structures of the eggs of Macrobiotus ripperi sp. nov. from Poland (PL.015) mounted in Hoyer’smedium(N–number of eggs/structures measured, RANGE refers to the smallest and the largest structure among all measured specimens; SD–standard deviation) CHARACTER N RANGE MEAN SD Egg bare diameter 30 70.7 – 82.1 76.6 3.0 Egg full diameter 30 89.2 – 108.8 99.4 4.4 Process height 90 8.8 – 16.7 13.2 1.7 Process base width 90 12.1 – 18.8 15.3 1.5 Process base/height ratio 90 83% – 177% 118% 18% Interprocess distance 90 3.6 – 9.0 5.6 1.2 Number of processes on the egg circumference 28 11 – 13 11.9 0.7 Stec et al. Zoological Letters (2021) 7:9 Page 29 of 45

Fig. 27 Macrobiotus ripperi sp. nov. from Poland (PL.015) – eggs seen in LCM: a–d – surface under ×1000 magnification of four different eggs; e–h – midsections of four different egg processes. The filled flat arrowhead indicates thickening perpendicular to the process base that divides the areola in the middle, the filled indented arrowhead indicates areas of the egg processes without a reticulation/labyrinthine layer, empty indented arrowheads indicate rings of the reticulation/labyrinthine layer in the bottom part of egg processes, and empty flat arrowheads indicate irregular collars around process bases. Scale bars in μm species exhibits secondary sexual dimorphism in the study. By the morphology of the animals and eggs, this form of clearly visible lateral gibbosities on hind legs in species can be differentiated from the following: males (Fig. 29b).  Macrobiotus pallarii: by lunulae IV being dentate (the lunulae are only faintly crenulated in M. DNA sequences and intraspecific genetic distances pallarii), by the absence of two lateral patches of dense granulation between legs III and IV (dense  18S rRNA sequences (GenBank: MT809074–9), 987 granulation patches between legs III and IV are bp long; 1 haplotype was found. present in M. pallarii; see Fig. 1), by the presence of  28S rRNA sequences (GenBank: MT809086–9), 716 a sparse granulation connecting the dense bp long; 1 haplotype was found. granulation patches between legs III and IV  ITS-2 sequences (GenBank: MT809100–2; extending posteriorly to the granulation on legs IV MT807929–32), 360 bp long; 2 haplotypes were (sparse granulation does not extend posteriorly to found, separated by a p-distance of 0.8%. the granulation on legs IV in M. pallarii; see Fig. 1)  COI sequences (GenBank: MT807933–5; and by and the absence of granulation on the egg MT809103–5) were 630 bp long; 3 haplotypes were processes tips (granulation is present in M. pallarii.; found, with p-distances ranging from 1.0% to 2.7%. character visible only under SEM).  Macrobiotus pseudopallarii sp. nov.: by lunulae Phenotypic differential diagnosis IV being dentate (the lunulae are only gently dentate By having the processes surrounded by 5–6 areolae, it in M. pseudopallarii sp. nov.), by the absence of resembles four other species of the Macrobiotus pallarii two lateral patches of dense granulation between complex out of which two are newly described in this legs III and IV (dense granulation patches between Stec et al. Zoological Letters (2021) 7:9 Page 30 of 45

Fig. 28 Macrobiotus ripperi sp. nov. from Poland (PL.015) – eggs seen with SEM: a – entire view of the egg; b–f – details of the egg surface between processes, areolation and egg processes. Filled flat arrowheads indicate thickening perpendicular to the process base, which divides the areola in the middle, empty flat arrowheads indicate irregular collar around process bases, and empty indented arrowheads indicate rings of the reticulation/labyrinthine layer in the bottom part of egg processes. Scale bars in μm

legs III and IV are present in M. pseudopallarii sp. all legs (granulation is not visible in M. nov.; see Fig. 1) and by and the absence of caymanensis), by the presence of a sparse dorsal granulation on the egg process tips (granulation granulation between legs III and IV (sparse present is in M. pseudopallarii sp. nov.; character granulation is absent or not visible in M. visible only under SEM). caymanensis) and by lunulae IV being dentate (the  Macrobiotus margoae sp. nov.: by having an oral lunulae are smooth in M. caymanensis). cavity armature that is well developed and composed of three bands of teeth visible with LM Genotypic differential diagnosis (the oral cavity armature is less developed, and the Interspecific genetic p-distances between M. ripperi sp. first band of teeth not visible with LM in M. nov. and other species of the M. pallarii complex are as margoae sp. nov), by the presence of sparse dorsal follows: granulation between legs III and IV (sparse granulation is absent in M. margoae sp. nov.; see  18S rRNA: 1.0–1.2% (1.1% on average), with the Fig. 1). most similar being Macrobiotus pallarii from Italy  Macrobiotus caymanensis: by having an oral cavity (MT809069–71) and Macrobiotus pseudopallarii sp. armature well developed and composed of three nov. from Montenegro (MT809065–6), and the bands of teeth visible with LCM (the oral cavity least similar being Macrobiotus margoae sp. nov. armature is less developed, and the first band of from the USA (MT809072–3). teeth is not visible with LCM in M. caymanensis),  28S rRNA: 2.1–2.5% (2.3% on average), with the by the presence of granulation visible with LCM in most similar being Macrobiotus margoae sp. nov. Stec et al. Zoological Letters (2021) 7:9 Page 31 of 45

Fig. 29 Macrobiotus ripperi sp. nov. from Poland (PL.015) – reproduction (LCM): a – spermatheca (seminal vesicle) filled with spermatozoa and visible in females freshly mounted in Hoyer’s medium; b – testis filled with sperm visible in a male freshly mounted in Hoyer’s medium. The flat arrowhead indicates the female spermathecae, the indented arrowhead indicates the testis, and arrows indicate gibbosities on the IV legs. Scale bars in μm

from the USA (MT809084–5), and the least similar Material examined: 101 animals and 36 eggs. Speci- being Macrobiotus pallarii from Italy (MT809081- mens were mounted on microscope slides in Hoyer’s 3). medium (90 animals + 29 eggs), fixed on SEM stubs (7+  ITS-2: 5.3–8.6% (6.8% on average), with the most 7), and processed for DNA sequencing (4+0). similar (to H2) being Macrobiotus pallarii from Italy Type locality: 35°35′7.84″N, 83°4′26.47″W; 1492 m (MT809094–6) and the least similar (to H1) being asl: United States: Great Smoky Mountains National Macrobiotus margoae sp. nov. H2 from the USA Park, Purchase Knob; Haywood County, North Carolina; (MT809097). moss on a tree trunk in the forest; coll. Nov 2018 by  COI: 18.6–22.7% (21.4% on average), with the most Nate Gross & Mackenzie McClay. similar (to H1) being Macrobiotus pallarii from Italy Type depositories: Holotype (slide US.057.07 with 1 (MT807924–6) and the least similar (to H3) being paratype), 95 paratypes (slides: US.057.02–06; SEM stub: Macrobiotus margoae sp. nov. from the USA 18.12) and 36 eggs (slides: US.057.01, 08–09; SEM stub: (MT807927–8). 18.12) are deposited at the Institute of Zoology and Bio- medical Research, Jagiellonian University, Gronostajowa Macrobiotus margoae Stec, Vecchi & Bartels, sp. 9, 30-387, Kraków, Poland. nov. Macrobiotus n. sp. 9 in [83, 84] Description of the new species Macrobiotus sp. in [85] Animals (measurements and statistics in Table 12): In Macrobiotus pallarii in [86–88] live animals, body almost transparent in smaller speci- Macrobiotus cf. pallarii US.057 [19] mens and whitish in larger animals; transparent after fix- Zoobank: urn:lsid:zoobank.org:act:ECD1BAC6-AC99- ation in Hoyer’s medium (Fig. 30). Eyes present in live 4991-B901-F0E5BDD92CC0 animals and after fixation in Hoyer’s medium. Small Etymology: This species is named in honor of PB's life round and oval cuticular pores (0.7–1.7 μm in diameter), partner, Margo Nottoli, who has shown tardigrade-like visible under both LCM and SEM, scattered randomly tolerance toward pressure, heat, and stress through 20 throughout the entire body (Figs. 31a–d, 32a–d). Patches years of her husband's research obsession. of fine granulation on the external surface of legs I–III Stec et al. Zoological Letters (2021) 7:9 Page 32 of 45

Table 12 Measurements [in μm] of selected morphological structures of individuals of Macrobiotus margoae sp. nov. from the USA (US.057) mounted in Hoyer’s medium (N–number of specimens/structures measured, RANGE refers to the smallest and largest structures among all measured specimens; SD–standard deviation) CHARACTER N RANGE MEAN SD Holotype μm pt μm pt μm pt μm pt Body length 30 239 – 443 690 – 1220 338 973 47 114 443 1220 Buccal tube Buccal tube length 30 29.4 – 41.9 – 34.8 – 2.9 – 36.3 – Stylet support insertion point 30 23.4 – 32.7 76.2 – 79.8 27.1 77.8 2.3 1.0 28.4 78.2 Buccal tube external width 30 3.1 – 6.3 9.5 – 16.8 4.9 14.1 0.7 1.7 6.1 16.8 Buccal tube internal width 30 2.8 – 4.9 8.6 – 15.7 3.8 11.0 0.5 1.4 4.2 11.6 Ventral lamina length 30 18.4 – 25.2 55.0 – 65.8 21.6 62.0 1.9 2.7 20.7 57.0 Placoid lengths Macroplacoid 1 30 6.3 – 11.8 20.0 – 29.9 9.0 25.8 1.2 2.2 10.1 27.8 Macroplacoid 2 30 4.1 – 7.1 11.1 – 19.7 5.8 16.6 0.9 2.1 7.1 19.6 Microplacoid 30 1.7 – 6.1 4.8 – 16.8 3.0 8.5 0.8 2.0 3.5 9.6 Macroplacoid row 30 11.7 – 20.3 40.0 – 55.9 16.0 45.9 2.0 3.4 20.3 55.9 Placoid row 30 16.0 – 24.7 52.1 – 68.0 19.8 56.8 2.1 3.1 24.7 68.0 Claw 1 heights External primary branch 25 6.3 – 11.1 20.4 – 30.6 8.5 24.2 0.9 2.0 11.1 30.6 External secondary branch 25 5.0 – 8.0 16.6 – 24.7 6.9 20.0 0.7 2.2 7.8 21.5 Internal primary branch 24 6.8 – 9.9 18.7 – 27.3 8.0 22.9 0.7 1.9 9.9 27.3 Internal secondary branch 22 5.5 – 7.5 14.8 – 21.4 6.5 18.7 0.5 1.8 7.5 20.7 Claw 2 heights External primary branch 24 7.7 – 10.4 20.3 – 28.6 8.9 25.5 0.7 2.0 10.1 27.8 External secondary branch 18 5.9 – 8.1 16.5 – 25.2 7.1 20.1 0.7 2.4 7.6 20.9 Internal primary branch 22 6.4 – 9.7 18.3 – 27.8 8.3 23.8 0.8 2.2 9.5 26.2 Internal secondary branch 20 5.4 – 7.7 15.3 – 22.2 6.7 19.3 0.6 2.1 7.5 20.7 Claw 3 heights External primary branch 22 7.2 – 10.3 21.0 – 27.6 8.9 25.2 0.9 2.0 10.0 27.5 External secondary branch 24 5.8 – 8.2 16.8 – 23.9 7.1 20.3 0.7 2.1 7.9 21.8 Internal primary branch 23 7.1 – 9.4 20.0 – 28.0 8.3 23.6 0.7 1.9 8.2 22.6 Internal secondary branch 18 5.4 – 8.0 14.4 – 22.3 6.5 18.3 0.8 2.2 ? ? Claw 4 heights Anterior primary branch 16 8.2 – 11.3 22.7 – 31.1 9.5 27.6 0.8 2.2 11.3 31.1 Anterior secondary branch 13 6.1 – 8.9 16.4 – 24.7 7.4 21.2 0.9 2.6 8.9 24.5 Posterior primary branch 14 8.4 – 11.5 24.5 – 32.6 9.9 28.8 0.8 2.6 11.5 31.7 Posterior secondary branch 18 6.4 – 9.0 20.1 – 26.7 7.6 22.0 0.8 1.7 ? ? as well as on the dorsal and dorsolateral sides of leg IV Lunulae on legs I–III smooth, whereas those on legs IV visible under LCM (Fig. 2, 31b, d) and SEM (Fig. 32b, d). clearly dentate (Fig. 33a–d). Dark areas under each claw on A pulvinus is present on the internal surface of legs I–III legs I–III faintly visible under LCM (Fig. 33a). Paired muscle (Figs. 31c, 32c). In addition to the typical patches of leg attachments and internal strengthening above them on legs granulation, other types of cuticular granulation are I–III were often visible under both LCM (Fig. 24a) and SEM, absent. whereas the horseshoe-shaped structure connecting anterior Claws slender, of the hufelandi type. Primary branches and posterior claws IV was visible only with LCM (Fig. 33b). with distinct accessory points, a long common tract, and an Mouth antero-ventral. Buccal apparatus of the Macro- evident stalk connecting the claw to the lunula (Fig. 33a–d). biotus type (Fig. 35a), with the ventral lamina and ten Stec et al. Zoological Letters (2021) 7:9 Page 33 of 45

Fig. 30 Macrobiotus margoae sp. nov. from the USA (US.057) – habitus, adult specimen in dorsoventral projection (holotype). Scale bar in μm peribuccal lamellae (Fig. 35a–b). The oral cavity arma- located within the posterior portion of the oral cavity, ture was composed of three bands of teeth, from which between the second band of teeth and the buccal tube only the second and third bands were always clearly vis- opening (Figs. 34b–c, 35a–b). The third band of teeth is ible under LCM (Fig. 34b–c), whereas the first band was divided into the dorsal and ventral portions. Under both only visible under SEM (Fig. 35a–b). The first band of LCM and SEM, the dorsal teeth are seen as three dis- teeth is composed of numerous small teeth visible as tinct transverse ridges, whereas the ventral teeth appear globular cones with SEM (Fig. 35a–b), arranged in sev- as two separate lateral transverse ridges, between which eral rows, and situated anteriorly in the oral cavity, just one large tooth (sometimes circular in LCM) is visible behind the bases of the peribuccal lamellae. The second (Figs. 34b–c, 35a–b). In SEM, only teeth of the dorsal band of teeth is situated between the ring fold and the portion in the third band have clearly indented margins third band of teeth and comprises 3–4 rows of teeth vis- (Fig. 35a). Pharyngeal bulb spherical, with triangular ible with LCM as granules (Fig. 34b–c) and with SEM as apophyses, two rod-shaped macroplacoids (2<1) and a cones (Fig. 35a–b) but larger than those in the first microplacoid positioned close to them (i.e., the distance band. The posterior row of teeth within the second band between the second macroplacoid and the microplacoid seems to comprise larger teeth than the previous anter- is shorter than the microplacoid length; Fig. 34d–e). The ior rows (Fig. 34b–c). The teeth of the third band are first macroplacoid is anteriorly narrowed and constricted

Fig. 31 Macrobiotus margoae sp. nov. from USA (US.057) – body and leg cuticle morphology seen with LCM: a – cuticle on the last body segment without caudal band of granulation; b – granulation on the external surface of leg III; c – internal surface of leg I with evident pulvinus; d – granulation on dorsal surface of leg IV. Scale bar in μm Stec et al. Zoological Letters (2021) 7:9 Page 34 of 45

Fig. 32 Macrobiotus margoae sp. nov. from USA (US.057) – body and leg cuticle morphology seen with SEM: a – cuticle on the last body segment without caudal band of granulation; b – granulation on the external surface of leg III; c – internal surface of leg III with evident pulvinus; d – granulation on dorsal surface of leg IV. Scale bar in μm.

Fig. 33 Macrobiotus margoae sp. nov. from USA (US.057) – claw morphology: a–b – claws II and IV seen with LCM; c–d – claws III and IV seen with SEM. Empty indented arrowhead indicates dark circular areas under lunulae on the first three pairs of legs, filled flat arrowhead indicates cuticular bar above muscle attachments, empty flat arrowhead indicates double muscle attachments under claws, arrow indicates horseshoe structure connecting the anterior and the posterior claws. Scale bars in μm Stec et al. Zoological Letters (2021) 7:9 Page 35 of 45

Fig. 34 Macrobiotus margoae sp. nov. from USA (US.057) – buccal apparatus seen with LCM: a – an entire buccal apparatus; b–c – the oral cavity armature, dorsal and ventral teeth, respectively; d–e – placoid morphology, dorsal and ventral placoids, respectively. Empty flat arrowheads indicate the second band of teeth, filled indented arrowheads indicate the third band of teeth, and empty indented arrowheads indicate central and subterminal constrictions in the first and second macroplacoid. Scale bars in μm in the middle, whereas the second has a subterminal smooth and not covered by granulation (Fig. 37c–f). The constriction (Fig. 34d–e). labyrinthine layer between the process walls was present Eggs (measurements and statistics in Table 13): Laid and usually visible only as small circular bubbles scat- freely, white, spherical with conical processes sur- tered randomly on the process (Fig. 36a–b). Rarely, these rounded by one row of areolae (Figs. 36a–h, 37a–f). In bubbles are almost not visible (Fig. 36c–d). The upper SEM, multiple rings of tight annulation on the entire part of the process is often elongated into short flexible process surface were visible (Fig. 37a–f) (annulation not apices that can be occasionally broken and that can visible in LCM because it was obscured by the eminent sometimes be bifurcated and have bubble-like structures labyrinthine layer). The upper parts of the processes are (Figs. 36e–h, 37c–f). The base of the processes extends

Fig. 35 Macrobiotus margoae sp. nov. from USA (US.057) – the oral cavity armature seen with SEM: a–b – the oral cavity armature of a single specimen seen in SEM from different angles showing dorsal and ventral portion, respectively. Filled flat arrowheads indicate the first band of teeth, empty flat arrowheads indicate the second band of teeth, and filled indented arrowheads indicate the third band of teeth. Scale bars in μm Stec et al. Zoological Letters (2021) 7:9 Page 36 of 45

Table 13 Measurements [in μm] of selected morphological structures of the eggs of Macrobiotus margoae sp. nov. from the USA (US.057) mounted in Hoyer’s medium (N–number of eggs/structures measured, RANGE refers to the smallest and largest structures among all measured specimens; SD–standard deviation) CHARACTER N RANGE MEAN SD Egg bare diameter 28 63.3 – 76.3 69.3 3.4 Egg full diameter 28 72.9 – 96.9 86.5 7.2 Process height 84 8.4 – 15.6 12.3 2.0 Process base width 84 8.2 – 16.5 12.5 1.5 Process base/height ratio 84 80% – 137% 104% 15% Interprocess distance 84 4.1 – 8.3 6.1 1.2 Number of processes on the egg circumference 28 10 – 14 12.1 1.0

into the six (only sometimes five) arms that form areolae two areolae by a narrow thickening perpendicular to the rims (Figs. 36a–d,37a–e). When this flattened part is process base (Figs. 36a–c, 37a–c). Areolae rims (walls) properly developed, the process base can be seen as hex- are usually thin and flat (Fig. 36a–d, 37a–e), with the agonal or pentagonal from the top view (Figs. 36a–d, labyrinthine layer inside the rims sometimes visible as 37a–b). Each process is surrounded by six (only some- bubbles under LCM (Fig. 36a–d). Areolae rims delimit times five) hexagonal areolae (Figs. 37a–d, a–e), which the areolae at the bases of processes, which forms an ir- are occasionally falsely subdivided in the middle into regular collar around process bases (Figs. 36a–d, 37a–d)

Fig. 36 Macrobiotus margoae sp. nov. from the USA (US.057) – eggs seen with LCM: a–d – surface under ×1000 magnification of four different eggs; e–h – midsections of four different egg processes. Filled flat arrowheads indicate thickenings perpendicular to the process base that divide the areola in the middle, empty indented arrowheads indicate bubbles within egg process walls, and empty flat arrowheads indicate irregular collar around process bases. Scale bars in μm Stec et al. Zoological Letters (2021) 7:9 Page 37 of 45

Fig. 37 Macrobiotus margoae sp. nov. from the USA (US.057) – eggs seen with SEM: a – entire view of the egg; b–f – details of the egg surface between processes, areolation and egg processes. Filled flat arrowheads indicate thickenings perpendicular to the process base that divide the areola in the middle, empty flat arrowheads indicate irregular collar around process bases. Scale bars in μm and makes the process bases penta- or hexagonal in DNA sequences and intraspecific genetic distances the top view (Figs. 36a–d, 37a–b). The areola surface has wrinkles that are faintly visible under LCM (Fig.  18S rRNA sequences (GenBank: MT809072–3), 987 36a–d) but clearly visible under SEM (Fig. 37a–e). bp long; 1 haplotype was found. Micropores are present within the areolae, but they  28S rRNA sequences (GenBank: MT809084–5), 715 are distributed only around the areolae rims and are bp long; 1 haplotype was found. usually absent in the central part of the areola (Fig.  ITS-2 sequences (GenBank: MT809097–9), 361 bp 37b–e). long; 2 haplotypes were found, separated by a p- distance of 0.8%.  COI sequences (GenBank: MT807927–8), 630 bp Reproduction long; 1 haplotype was found. The species is dioecious. Spermathecae in females as well as testes in males have been found to be filled with Phenotypic differential diagnosis spermatozoa, clearly visible under PCM up to 24 hours By having the processes surrounded by 5–6 areolae, it after mounting in Hoyer’s medium (Fig. 38a–b). The resembles four other species of the Macrobiotus pallarii species exhibits secondary sexual dimorphism in the complex out of which two are newly described in this form of clearly visible lateral gibbosities on hind legs in study. By the morphology of the animals and eggs, this males (Fig. 38b). species can be differentiated from the following: Stec et al. Zoological Letters (2021) 7:9 Page 38 of 45

Fig. 38 Macrobiotus margoae sp. nov. from the USA (US.057) – reproduction (LCM): a – spermatheca (seminal vesicle) filled with spermatozoa and visible in females freshly mounted in Hoyer’s medium; b – testis filled with sperm visible in a male freshly mounted in Hoyer’s medium. The flat arrowhead indicates the female spermathecae, the indented arrowhead indicates the testis, and the arrow indicates gibbosity on the IV leg. Scale bars in μm

 Macrobiotus pallarii: by a weakly developed oral of teeth not visible under LM (the oral cavity cavity armature, with the first band of teeth not armature is well developed, and the first band of visible under LCM (the oral cavity armature is well teeth is visible under LM in M. pseudopallarii sp. developed, and the first band of teeth is visible nov.), by dentate lunulae IV (lunulae are gently under LCM in M. pallarii), by dentate lunulae IV dentate in M. pseudopallarii sp. nov.), by the (lunulae are faintly crenulate in M. pallarii), by the absence of two lateral patches of dense absence of two lateral patches of dense granulation granulation between legs III and IV (the dense between legs III and IV (dense granulation patches granulation patches between legs III and IV are between legs III and IV are present in M. pallarii; present in M. pseudopallarii sp. nov.;seeFig.1), see Fig. 1), by the absence of a sparse dorsal by the absence of sparse dorsal granulation granulation between legs III and IV (sparse between legs III and IV (the sparse granulation is granulation is present in M. pallarii; see Fig. 1), by a present in M. pseudopallarii sp. nov.;seeFig.1), lower placoid row pt value (51.1–60.6 in M. margoae by a lower placoid row pt value (51.1–60.6 in M. sp. nov. vs. 61.3–75.6 in M. pallarii), by the margoae sp. nov. vs. 65.0–75.1 in M. presence of small circular bubbles scattered pseudopallarii sp. nov.), by the presence of small randomly within the egg process walls (meshes are circular bubbles scattered randomly within the present within the entire process walls in M. process walls (meshes are present within the walls pallarii), and by the absence of granulation on the of the entire process in M. pseudopallarii sp. egg process tips (granulation is present in M. nov.), and by and the absence of granulation on pallarii; character visible only under SEM). the egg processes tips (granulation is present in  Macrobiotus pseudopallarii sp. nov.: by a weakly M. pseudopallarii sp. nov.;charactervisibleonly developed oral cavity armature, with the first band under SEM). Stec et al. Zoological Letters (2021) 7:9 Page 39 of 45

 Macrobiotus ripperi sp. nov.: by a weakly 1 Egg processes surrounded by 5–6 areolae...... 2 developed oral cavity armature, with the first band - Egg processes surrounded by 11–12 areolae M. of teeth not visible under LCM (the oral cavity ragonesei Binda et al., 2001 [26] armature is well developed, and the first band of 2(1) Granulation on all legs present...... 3 teeth is visible under LCM in M. ripperi sp. nov.) - Granulation on all legs absent or not visible with and by the absence of sparse dorsal granulation LCM M. caymanensis Meyer, 2011 [28] between legs III and IV (sparse granulation is 3(2) Two lateral patches of dense granulation between present in M. ripperi sp. nov.; see Fig. 1). legs III and IV present...... 4  Macrobiotus caymanensis: by the presence of - Two lateral patches of dense granulation between legs granulation visible under LCM on all legs III and IV absent...... 5 (granulation is not visible in M. caymanensis) and by 4(3) Lunules IV faintly crenulate, sparse granulation dentate lunulae IV (lunulae are smooth in M. connecting the dense granulation patches between legs caymanensis). III and IV not extending posteriorly...... M. pallarii Maucci, 1954 [22] - Lunules IV dentate, sparse granulation connecting the Genotypic differential diagnosis dense granulation patches between legs III and IV Interspecific genetic p-distances between M. margoae extending posteriorly to the granulation on legs sp. nov. and other species of the M. pallarii complex IV...... M. pseudopallarii sp. nov. are as follows: 5(3) Sparse dorsal granulation between legs III and IV present...... M. ripperi sp. nov.  18S rRNA: 1.2–1.4% (1.2% on average), with the - Sparse dorsal granulation between legs III and IV most similar being Macrobiotus pallarii from Italy absent...... M. margoae sp. nov. (MT809069–71) and Macrobiotus ripperi sp. nov. from Poland and Finland (MT809074–6), and the Discussion least similar being Macrobiotus pseudopallarii sp. Macrobiotus pallarii was described from Italy in 1954, nov. from Montenegro (MT809067–8), and it has been reported from Europe and North Amer-  28S rRNA: 2.1–2.7% (2.3% on average), with the ica [25, 89, 90]. We recovered a population of this spe- most similar being Macrobiotus ripperi sp. nov. cies from the type locality (Silvana Mansio, Cosenza, from Poland and Finland (MT809086–9), and the Italy) in an environment matching the original species least similar being Macrobiotus pallarii from Italy description (moss on rock on the ground in a sparse for- (MT809081–3). est [22]). The assignment of population IT.337 to M.  ITS-2: 6.1–8.6% (7.8% on average), with the most pallarii was confirmed by a comparison with the type similar (to H2) being Macrobiotus pallarii from Italy series from the Maucci collection (Natural History Mu- (MT809094–6) and the least similar (to H2) being seum of Verona). The analysis of the topotypes of M. Macrobiotus ripperi sp. nov. H1 from Poland and pallarii, with four distinct populations (that would have Finland (MT809100–3). been attributed to the same species prior to this study),  COI: 21.1–22.7% (21.4% on average), with the most showed that four pseudocryptic species can be distin- similar being Macrobiotus pallarii from Italy guished from morphological, morphometric, and genetic (MT807924–6) and the least similar being viewpoints. Due to the presence of these morphological Macrobiotus ripperi sp. nov. H3 from Finland and genetic differences, we described them as three new (MT807930–2). species (see the Taxonomic Account above).

Dichotomous diagnostic key to species of the Species delineation in the Macrobiotus pallarii complex Macrobiotus pallarii complex Congruent with the results of species delimitation of dif- To facilitate species identification, we provide a dichot- ferent populations of the Paramacrobiotus areolatus omous key to the valid species of the M. pallarii com- group by [7], the COI showed higher divergence than plex. When differentiating these species, we stress the ITS-2, and a strikingly similar pattern was found for a need for analyzing the largest possible number of indi- pair of closely related P. areolatus group populations viduals and eggs, as some characters, such as egg process (IT.048 and PT.006 in [7]) with contrasting species de- reticulation, show intraspecific variability (see the Taxo- limitation results based on the two analyzed markers nomic Account above), and some more conservative (distinct species according to COI, same species accord- characters, such as dorso-caudal granulation, may not al- ing to ITS-2). Similar differences in species delimitation ways be easily detected due to suboptimal orientation of based on COI and ITS-2 markers were obtained for specimens on slides or/and microscope quality. some species in the genus Milnesium, although in Stec et al. Zoological Letters (2021) 7:9 Page 40 of 45

contrast to the present study, the precedence was given from the Andaman Islands in the Indian Ocean, is to ITS-2, as no phenotypic differences between units de- currently assigned to the Macrobiotus polyopus group lineated with COI were detected [12, 91]. Thus, it seems [93]. However, because of its similarity with the spe- that there is no simple rule to decide which genetic cies of the M. pallarii complex, it would not be sur- marker is better suited for species delineation in tardi- prising if after a reanalysis of the type material, the grades. Rather, both phenotypic and genetic traits should species is moved to the latter complex. Additionally, be used in tandem in taxonomic analyses. M. caymanensis wasoriginallydescribedasamember of the M. polyopus group, which suggests that both Species diversity in the Macrobiotus pallarii complex groups have not been sufficiently differentiated and Macrobiotus pallarii, M. ragonesei, M. caymanensis, and doubts about the assignment to one or the other the three new species form the M. pallarii complex (de- group are possible. In our opinion, the M. polyopus fined by [19] as having cone-shaped egg processes sepa- group can be differentiated from the M. pallarii com- rated by one row of areolation). The identification of plex by the morphology of egg processes and areola pseudocryptic species in the M. pallarii complex re- walls (rims) (Fig. 39a). Specifically, process base width opens the question about the identity of M. aviglianae, and interprocess distances were similar in the M. which has been synonymised with M. pallarii by [24]. polyopus group (Fig. 39b), whereas process bases were The analysis of the M. aviglianae-type material (slides much wider than the distance between neighboring CT1954, CT1955, CT1966, CT4051 and CT4485 of the processes in the M. pallarii complex (Fig. 37b). Maucci collection) showed that it was not in good con- Moreover, areolae are always delimited by high and dition. Thus, it is not possible to verify whether M. thin walls in the M. polyopus group (usually nearly aviglianae does not differ from M. pallarii in any half the process height, Fig. 39c–d), whereas areolae subtle traits, such as in the pattern of dorso-caudal walls are low and often thick, with the labyrinthine granulation or granulation on the tips of egg pro- layer visible in LCM, in the M. pallarii complex (Fig. cesses. Moreover, it is also not possible to test 37c–d). Thus, currently, there are six confirmed spe- whether M. aviglianae represents an independent cies in the M. pallarii complex, with further potential phyletic lineage or if it falls within the intraspecific members: M. insularis (pending the verification of the genetic variability of M. pallarii. Thus, only the dis- type material) and M. aviglianae (pending an examin- covery of a new population of M. aviglianae in its ation of a topotypic population). However, the num- type locality will allow for a proper test of its syn- ber of species constituting the complex is likely to be onymy with M. pallarii. The morphometric characters much higher, as has been shown in other eutardi- of the M. caymanensis type overlap with those of the grade groups when tools of integrative taxonomy and newly described M. margoae sp. nov.,withwhichit large-scale sampling have been employed, for ex- also shares a similar appearance of the egg process ample, in the genus Milnesium [94], the genera wall. Thus, it is probable that these two species are Astatumen and Platicrista [95], the Ramazzottius phylogenetically close; however, it will be possible to oberhaeuseri complex [38], the genus Macrobiotus test this hypothesis only after genetic data for M. cay- (e.g., [19]), the Paramacrobiotus richtersi and areola- manensis are obtained. For M. ragonesei, detailed tus complexes ([5, 7], respectively), or in the genus morphometric data are not available, precluding its [6, 13]. inclusion in the PCA, but based on morphology alone, it is clear that the species deviates from all Biogeography of the Macrobiotus pallarii complex other known members of the complex by a much Species of the Macrobiotus pallarii complex are not higher number of areolae around each egg process commonly found, but the complex has a wide, pos- (11–12 vs.5–6 in the remaining species). Macrobiotus sibly global distribution. Although the majority of re- ragonesei was collected only once in the type locality cords are concentrated in Europe (Finland, Greece, in the Democratic Republic of Congo (Central Africa); Hungary, Italy, Montenegro, Norway, Poland, Republic however, it is possible that the population collected of Bulgaria, Russian Federation, Turkey, Yugoslavia and identified as Macrobiotus cf. pallarii by [92]from [25, 82, 96–98]), there are also reports from Asia Swaziland (South Africa) represents M. ragonesei or a (North Korea, Russian Federation [25]), North Amer- related species because eggs from the Congolese ica (United States, Cayman Islands [28, 86–88]), and population resembled those of M. ragonesei (“pro- Africa (Democratic Republic of the Congo, Swaziland cesses are rounded rather than tapering, and the [27, 92]). In light of our findings, all these records ex- number of areolae surrounding them is ten to twelve cept the type localities should be considered dubious rather than eight or nine” [92]). Another species, and be designated as species of the Macrobiotus pal- Macrobiotus insularis Pilato, 2006 [93], known only larii complex, i.e., as Macrobiotus aff. pallarii spp., Stec et al. Zoological Letters (2021) 7:9 Page 41 of 45

Fig. 39 Macrobiotus aff. polyopus species from Papua New Guinea – eggs seen with SEM: a – entire egg; b–d – details of the egg surface between the processes, areolation and egg processes. Scale bars in μm keeping open the possibility they could represent yet requirements despite being very closely related. The M. undescribed new species. pallarii complex also seems to be ecologically and physiologically diversified, as M. ripperi sp. nov. per- The importance of disentangling pseudocryptic species forms very well in laboratory culture (D.S. and M.V. per- complexes sonal observation), whereas the other examined species Thanks to the use of integrative taxonomy, pseudocryp- (M. pallarii, M. pseudopallarii sp. nov., M. margoae sp. tic and cryptic tardigrade species complexes are being nov.), while able to reproduce in culture, they did not detected with increasing frequency (chronologically [6, thrive. Whether these differences in reproductive success 7, 11, 13, 14, 19, 38, 99–103]). Other than for purely under uniform laboratory conditions are a result of nat- taxonomic purposes, acknowledging and discerning such ural variability in the physiology or different levels of entities may significantly impact studies concerning bio- alignment of the ecology of these species and laboratory diversity, biogeography, ecology or physiology. Specific- conditions (i.e., the similarity of laboratory conditions, ally, biodiversity studies and taxonomic checklists need such as temperature, fodder type or photoperiod, to the precise taxonomic identifications to be comparable conditions to which the species is adapted in nature) has across different geographical areas. However, as noted, yet to be tested. Nevertheless, in both cases, species e.g., by [82], numerous identifications cannot be con- identity, despite the striking morphological similarity, is firmed due to the presence of (pseudo) cryptic species an important variable in the equation and cannot be complexes. Furthermore, biogeographical studies sup- ignored. ported by molecular data on tardigrades are still scarce. Differences in ecological and physiological traits in spe- In one of them, Gąsiorek et al. [104] showed evidence cies that exhibit very little morphological differentiation suggesting allopatric speciation in a complex of tardi- have also been demonstrated for cryptic species com- grades (Echiniscus virginicus complex) that are almost plexes in other taxa (e.g., bryozoans, diatoms and butter- indistinguishable morphologically but genetically diver- flies [106–108]). The presence of cryptic species that show gent, underlining the need for genetic verification of physiological and life history divergence can also influence faunistic records to draw sound biogeographic conclu- laboratory studies when the adopted model species belong sions. Regarding ecology, for example, Faurby et al. [105] to one of those complexes. For example, studies on the found that Echiniscoides species have different geograph- model sea squirt Ciona intestinalis (Linnaeus, ical distributions and thus also various ecological 1767 )[109] assumed that all worldwide natural Stec et al. Zoological Letters (2021) 7:9 Page 42 of 45

populations belong to a single species [110]. However, it account to avoid incongruent results stemming from taxo- was later demonstrated that cryptic species that produce nomic misidentifications. sterile hybrid offspring are present [111]. Acknowledg- ment of the presence of cryptic species in C. intestinalis Supplementary Information turned out to be essential for experimental design and The online version contains supplementary material available at https://doi. org/10.1186/s40851-021-00176-w. data management [110], as, for example, new laboratory strains could not be obtained by crossing the cryptic spe- Additional file 1: SM.01. Saturation plots for COI and ITS markers. cies. There are also similar examples concerning tardi- Additional file 2: SM.02. MrBayes analysis input file with the alignment. grades. Specifically, individuals attributed to Additional file 3: SM.03. The p-distances between species of the Paramacrobiotus richtersi were used by [112]totestthe Macrobiotus pallarii complex. effects of a lower earth orbit environment on tardigrade Additional file 4: SM.04a. R data for morphometric analysis. physiology and reproduction. However, it was later re- Additional file 5: SM.04b. R script for morphometric analysis. vealed that individuals of that population belong to a dif- Additional file 6: SM.05. Raw morphometric data for Macrobiotus ferent cryptic species of the P. richtersi complex, pallarii Maucci, 1954 from the topotypic population (IT.337). Paramacrobiotus spatialis Guidetti et al., 2019 [5]. As spe- Additional file 7: SM.06. Raw morphometric data for Macrobiotus cies in the P. richtersi complex show different geographic pseudopallarii sp. nov. from Montenegro (ME.007, type population). distributions [5] and are likely to have different environ- Additional file 8: SM.07. Raw morphometric data for Macrobiotus ripperi sp. nov. from Poland (PL.015, type population). mental requirements and tolerances, their misidentifica- Additional file 9: SM.08. Raw morphometric data for Macrobiotus tion (due to the recognition of the species complex years ripperi sp. nov. from Finland (FI.066, additional population). later) could affect the replicability of the 2011 study if the Additional file 10: SM.09. Raw morphometric data for Macrobiotus experiment was replicated with another species of the margoae sp. nov. from USA (US.057, type population). complex identified as “P. richtersi”.Similarly,moretardi- Additional file 11: SM.10. Results of PCA randomization tests. grade species used in experimental studies were found to belong to cryptic (or pseudocryptic) species complexes Acknowledgments We are especially grateful to our colleagues Piotr Gąsiorek, Aleksandra and misidentified for the nominal species of the complex, Rysiewska, Nate Gross, Mackenzie McClay, Francesco Squillace and Anna for example Hypsibius exemplaris was misidentified as H. Millard for collecting and sharing the samples that made this study possible. dujardini [113], Milnesium inceptum was misidentified as We also thank Roberto Guidetti and the Civic Museum of Natural History of Verona for making the Maucci collection available and for sending M. tardigradum [114]andRichtersius sp. 4 was misidenti- photomicrographs of paratypes of M. pallarii and M. aviglianae. Sample IT.337 fied as R. coronifer [6]. Importantly, while discrepancies was collected under a sampling permit from Ente Parco Nazionale della Sila stemming from results obtained with different (pseudo) (Prot.N.0007433/2019) issued to MV. cryptic species could be a problem when not recognized, Trial registration such species may also provide a useful tool to investi- Not applicable. gate tardigrade speciation and how isolation between such ’ morphologically similar species is maintained by using the Authors contributions DS conceptualized and designed the work, collected and analyzed tools of experimental taxonomy, such as interpopulation morphological and molecular data, acquired images, prepared figures, crosses [7] and mate choice assays, as well as omics interpreted results, and drafted the work. MV conceptualized and designed techniques. the work, analyzed morphological and molecular data, prepared figures, interpreted the results and drafted the work. MD collected morphometric data. PB and SC interpreted results. ŁM conceptualized and designed the work, interpreted the results and drafted the work. All authors read and Conclusions approved the final manuscript.

Integrative taxonomy revealed that multiple pseudocryptic Funding species, each probably with a limited geographic distribu- The study was supported by the Preludium programme of the Polish tion, are present under what was once considered a single National Science Centre (grant no. 2018/31/N/NZ8/03096 to DS, supervised “ ” by ŁM) and by the European Commission’s (FP 6) Integrated Infrastructure cosmopolitan species, Macrobiotus pallarii .Speciesof Initiative SYNTHESYS (grant no. DK-TAF-4387 to ŁM). Some of the analyses the Macrobiotus pallarii complex can be differentiated were carried out with equipment purchased from the Sonata Bis programme principally by dorsal granulation and egg ornamentation of the Polish National Science Centre (grant no. 2016/22/E/NZ8/00417 to ŁM). During this study, DS was a beneficiary of a National Science Centre but also statistically by morphometric traits. Other than scholarship to support doctoral research (no. 2019/32/T/NZ8/00348). SC and for purely taxonomic reasons, disentangling tardigrade MV are funded by the Academy of Finland (Fellowship #314219 to SC). cryptic and pseudocryptic species complexes is fundamen- Open-access publication of this article was funded by the BioS Priority Re- search Area under the program “Excellence Initiative – Research University” tal for the proper interpretation of biogeographical and at Jagiellonian University in Kraków, Poland. ecological studies and the replicability of experimental works. Due to the rising popularity of tardigrade species Availability of data and materials – All data generated or analyzed during this study are included in this as laboratory models (e.g., [17, 115 124]), the presence of published article [and its supplementary information files]. Genetic data are cryptic or pseudocryptic complexes must be taken into deposited in GenBank, NCBI. Stec et al. Zoological Letters (2021) 7:9 Page 43 of 45

Declarations 15. Morek W, Ciosek JA, Michalczyk Ł. Description of Milnesium pentapapillatum sp. nov., with an amendment of the diagnosis of the order Apochela and Ethics approval and consent to participate abolition of the class Apotardigrada (Tardigrada). Zool Anz. 2020;288:107–17. Not applicable 16. Grothman GT, Johansson C, Chilton G, Kagoshima H, Tsujimoto M, Suzuki AC. Gilbert Rahm and the status of Mesotardigrada Rahm, 1937. Zool Sci. 2017;34(1):5–10. Consent for publication 17. Bryndová M, Stec D, Schill RO, Michalczyk Ł, Devetter M. Dietary preferences Not applicable. and diet effects on life-history traits of tardigrades. Zool J Linnean Soc. 2020;188(3):865–77. 18. Schultze CAS. Macrobiotus Hufelandii, animal e crustaceorum classe novum, Competing interests reviviscendi post diuturnam asphyxiam et ariditatem potens. Etc. 8, C. The authors declare that they have no competing interests. Curths; 1834. 19. Stec D, Vecchi M, Calhim S, Michalczyk Ł. 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